Treatment of parkinson&#39;s disease via administration of GLI-1 protein

ABSTRACT

Described herein are compositions and methods for the treatment of Parkinson&#39;s disease (PD) and/or to protect dopaminergic nigrostriatal neuronal cell bodies from 6-OHDA-induced neurotoxicity in a mammal. In various embodiments of the invention, the dopaminergic neuron differentiation factor sonic hedgehog (Shh) and/or its downstream transcription factor target Gli-1 are used in connection with gene therapeutic techniques or direct peptide injection for the aforementioned indications. Kits useful in practicing the inventive method are also disclosed, as are animal models useful for studying various neurodegenerative conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/572,397, filed Jan. 4, 2008, currently pending, which is the nationalphase of International Patent Application No. PCT/US05/29192, filed Aug.11, 2005, which designated the U.S. and was published under PCT Article21(2) in English and also included a claim of priority under 35 U.S.C.119(e) to U.S. provisional patent application No. 60/600,629, filed Aug.11, 2004.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant Nos.R01NS42893 and R01NS44556 awarded by National Institutes of Health.

FIELD OF THE INVENTION

The invention relates to the field of neurodegenerative disorders, and,in particular embodiments, to Parkinson's disease.

BACKGROUND OF THE INVENTION

Treatments for Parkinson's disease (PD), although effective, do not haltthe progressive loss of substantia nigra dopaminergic neurons.Eventually, clinical symptoms become resistant to treatments relying onthe integrity of nigrostriatal neurons, such as L-DOPA [S. Mandel etal., Neuroprotective strategies in Parkinson's disease: an update onprogress, CNS Drugs, 17:729-762 (2003)]. Preserving viable nigrostriatalneurons would delay disease progression and thus prolong treatments'efficacy [J. H. Kordower et al, Neurodegeneration prevented bylentiviral vector delivery of GDNF in primate models of Parkinson'sdisease, Science, 290:767-773 (2000); D. Kirik et al., Long-termrAAV-mediated gene transfer of GDNF in the rat Parkinson's model:intrastriatal but not intranigral transduction promotes functionalregeneration in the lesioned nigrostriatal system, J. Neurosci.,20:4686-4700 (2000); D. L. Choi-Lundberg et al, Dopaminergic neuronsprotected from degeneration by GDNF gene therapy, Science. 275:838-841(1997); M. G. Castro et al., Gene therapy for Parkinson's disease:recent achievements and remaining challenges, Histol. Histopathol.,16:1225-1238 (2001)]. Glia1-cell-derived neurotrophic factor (GDNF)protects nigral dopaminergic cell bodies and their striatal axonterminals from in vitro and in vivo neurotoxicity induced by6-hydroxydopamine (6-OHDA) [D. L. Choi-Lundberg et al., Dopaminergicneurons protected from degeneration by GDNF gene therapy, Science,275:838-841 (1997)4], MPTP [J. H. Kordower et al., Neurodegenerationprevented by lentiviral vector delivery of GDNF in primate models ofParkinson's disease, Science, 290:767-773 (2000)], or metamphetamine [W.A. Cass, GDNF selectively protects dopamine neurons over serotoninneurons against the neurotoxic effects of methamphetamine, J. Neurosci.,16:8132-8139 (1996)], and possibly also in PD patients [S. S. Gill etal, Direct brain infusion of glial cell line-derived neurotrophic factorin Parkinson disease, Nat. Med., 9:589-595 (2003)].

GDNF has been delivered into the brain using adenovirus (RAd)-,adeno-associated virus-, herpes simplex virus type 1 (HSV-1)-, orlentiviral-derived vectors or by direct peptide injection [M. G. Castroet al., Gene therapy for Parkinson's disease: recent achievements andremaining challenges, Histol. Histopathol., 16:1225-1238 (2001); S. S.Gill et al., Direct brain infusion of glial cell line-derivedneurotrophic factor in Parkinson disease, Nat. Med., 9:589-595 (2003);A. Bjorklund et at, Towards a neuroprotective gene therapy forParkinson's disease: use of adenovirus, AAV and lentivirus vectors forgene transfer of GDNF to the nigrostriatal system in the rat Parkinsonmodel, Brain Res. 886:82-98 (2000); E. A. Burton et al., Gene therapyprogress and prospects: Parkinson's disease, Gene Ther., 10:1721-1727(2003)]. Despite its neuroprotective actions, GDNF can have untowardeffects (i.e., reduction of tyrosine hydroxylase mRNA in nigrostriatalneurons, aberrant morphologies of striatal tyrosinehydroxylase-immunoreactive axons, and increased cell death followingexperimental stroke) [B. Georgievska et al., Aberrant sprouting anddownregulation of tyrosine hydroxylase in lesioned nigrostriataldopamine neurons induced by long-lasting overexpression of glial cellline derived neurotrophic factor in the striatum by lentiviral genetransfer, Exp. Neurol., 177:461-474 (2002); A. Arvidsson et al.,Elevated GDNF levels following viral vector-mediated gene transfer canincrease neuronal death after stroke in rats, Neurobiol. Dis.,14:542-556 (2003); C. Rosenblad et al., Long-term striataloverexpression of GDNF selectively downregulates tyrosine hydroxylase inthe intact nigrostriatal dopamine system, Eur. J. Neurosci., 17:260-270(2003)].

The disclosures of all documents referred to throughout this applicationare incorporated herein by reference. The foregoing examples of therelated art and limitations related therewith are intended to beillustrative and not exclusive. Other limitations of the related artwill become apparent to those of skill in the art upon a reading of thespecification and a study of the drawings.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

One embodiment of the present invention includes a method for treatingParkinson's disease in a mammal, including providing a quantity of aviral vector expressing ShhN and administering a therapeuticallyeffective amount of the quantity of the viral vector to the mammal.

Another embodiment of the present invention includes a method fortreating Parkinson's disease in a mammal, including providing a quantityof a viral vector expressing Gli-1 and administering a therapeuticallyeffective amount of the quantity of the viral vector to the mammal.

Another embodiment of the present invention includes a method forprotecting dopaminergic nigrostriatal neuronal cell bodies from6-OHDA-induced neurotoxicity in a mammal, including providing a quantityof a viral vector expressing ShhN and administering a therapeuticallyeffective amount of the quantity of the viral vector to the mammal.

Another embodiment of the present invention includes a method forprotecting dopaminergic nigrostriatal neuronal cell bodies from6-OHDA-induced neurotoxicity in a mammal, including providing a quantityof a viral vector expressing Gli-1 and administering a therapeuticallyeffective amount of the quantity of the viral vector to the mammal.

Another embodiment of the present invention includes a method fortreating PD in a mammal, including providing a composition comprising aShhN protein, a Gli-1 protein, or both, and administering atherapeutically effective amount of the composition to the mammal.

Another embodiment of the present invention includes a method forprotecting dopaminergic nigrostriatal neuronal cell bodies from6-OHDA-induced neurotoxicity in a mammal, including providing acomposition comprising a ShhN protein, a Gli-1 protein, or both, andadministering a therapeutically effective amount of the composition tothe mammal.

Another embodiment of the present invention includes a kit, including acomposition comprising a viral vector expressing ShhN, and instructionsfor its use for treating PD in a mammal.

Another embodiment of the present invention includes a kit, including acomposition comprising a viral vector expressing and instructions forits use for treating PD in a mammal.

Another embodiment of the present invention includes a kit, including acomposition comprising ShhN protein, Gli-1 protein, or both, andinstructions for its use for treating PD in a mammal.

Another embodiment of the present invention includes an in vivo model ofnigrostriatal neurodegeneration, comprising a non-human mammal thatcarries in at least a portion of the cells of its brain at least oneexogenous ShhN DNA encoding a ShhN peptide.

Another embodiment of the present invention includes an in vivo model ofnigrostriatal neurodegeneration, comprising a non-human mammal thatcarries in at least a portion of the cells of its brain at least oneexogenous Gli-1 DNA encoding a Gli-1 peptide.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than restrictive.

FIG. 1 illustrates the genomic structures of RAd-GDNF, RAd-ShhN, andRAd-Gli-1, in accordance with an embodiment of the present invention.Recombinant adenoviruses (RAd) were generated by homologousrecombination after cotransfection into 293 cells of a shuttleexpression plasmid encoding ShhN, Gli-1, or Nurr-1 together with the Ad5genomic plasmid pJM17. The shuttle plasmid contained adenoviral DNAsequences encoding the left-end replication origin/packaging elementsand the overlap-recombination region. Restriction patterns of adenoviralvectors digested with HindIII confirmed the presence of the transgenes.Identity of the transgenes was confirmed by Southern blot hybridization,using specific DIG-labeled probes. (a) The schematic structure of thenew vectors described herein. (b, c) The characterization of RAd-GDNF,with (b) the restriction analysis and (c) the Southern blothybridization indicating the presence of the expected transgene band(1.4 kb), in lanes 1 and 2. In b and c the lane numbers indicate 1, theshuttle vector used to construct the recombinant virus, pALGDNF (aspositive control); 2, RAd-GDNF; 3, the Ad5 genomic plasmid pJM17 (asnegative control); and 4, molecular weight markers. (d, e) Thecharacterization of RAd-ShhN, with (d) the restriction analysis and (e)the Southern blot hybridization indicating the presence of the bandcontaining the transgene ShhN (0.9 kb), in lanes 1 and 2. In d and e thelane numbers indicate 1, RAd-ShhN; 2, the shuttle plasmid pALShhN (aspositive control); and 3, molecular weight markers. (f, g) Thecharacterization of RAd-Gli-1, with (f) the restriction analysis and (g)the Southern blot hybridization indicating the presence of the bandcontaining the transgene Gil-1, of approx 4.0 kb, in lanes 1 and 3. In fand g the lane numbers indicate MW, molecular weight markers; 1,RAd-Gli-1; 2, pJM17 (as negative control); and 3, pALGli-1 (as positivecontrol). The construction of Nurr-1 followed identical principles butis not illustrated.

FIG. 2 illustrates that ShhN is produced and released by BHK or glialcells infected with RAd-ShhN in accordance with an embodiment of thepresent invention. The presence of ShhN in the supernatant of BHK cellsinfected with RAd-ShhN is shown in (a, b) a Western blot analysis, (c) adot blot, and (d) an ELISA. Electrophoretic separation of conditionedmedium (CM) from RAd-infected BHK cells on 10% Nu-PAGE gel is shown in(a). Lanes 1-4 contain 25-fold-concentrated samples from the differentCM: (1) serum-free (SF) medium, (2) CM mock. (3) CM LacZ, (4) CM ShhN.MW corresponds to molecular weight standards. Western blot analysis (b)confirmed that ShhN (approx 20 kDa) was released into culture mediumafter RAd-ShhN infection. Dot-blot analysis is illustrated in (c): 200μl of 50% conditioned medium from RAd-infected BHK cells wasimmunoreacted with anti-ShhN antibodies. This assay demonstrated thatShhN was detected only in the conditioned medium from BHK cells infectedwith RAd-ShhN. This assay was carried out using conditioned medium atthe same concentration used for bioactivity assays. Conditioned mediumfrom BHK cells infected with increasing m.o.i. of 0-1000, and assayedfor ShhN using an ELISA, is shown in (d). Two way ANOVA: m.o.i.F327=1.586, P≧0.05. RAd F127=72.423, P≦0.001. RAd*m.o.i. F327=3.231,P≦0.05 (+). Dunnett t (two-tailed) post hoc test for RAd effects:RAd-CMV-ShhN vs mock, P≦0.001, but RAd-35 vs mock, P≧0.05. Dunnett t(two-tailed) post hoc test for RAd*m.o.i. interaction: RAd-ShhN 100 vsmock, P≦0.05 (++); RAd-ShhN 300 vs mock (+++), and RAd-ShhN 1000 vsmock, P≦0.01 (++). The other possible RAd*m.o.i. combinations were notsignificant compared to mock-infected cultures. This illustrates thatthe release of ShhN into the medium, following RAd-ShhN-infection of BHKcells, increased proportionally to RAd-ShhN m.o.i. and reached its peakat 300 m.o.i.; this m.o.i. was selected for production of theconditioned media for further bioactivity studies. In addition, to testwhether ShhN would also be produced and released from rodent glialcells, primary cultures of glial cells were infected with RAd-ShhN. Thecontrol cells are illustrated in (e) and infected cells expressing ShhNare shown in (f). Release of ShhN into the supernatant, analysis by dotblot, is shown in (g). Two hundred microliters of 50% conditioned mediumfrom mock or RAd-infected glial cells was immunoreacted with a specificanti-ShhN antibody. This assay demonstrated that ShhN was released onlyinto the conditioned medium originating from glial cells infected withRAd-ShhN.

FIG. 3 illustrates that RAd-ShhN increases the survival of dopaminergicneurons (TH+ neurons) in ventral-mesencephalic (VM) cultures, inaccordance with an embodiment of the present invention. E14 VM cultureswere incubated for four days with (a, b) CM from mock-infected BHK cellsor CM from BHK cells infected with (c, d) RAd-35 or (e, RAd-ShhN. Inparallel cultures cells were pretreated with the anti-ShhN blockingantibody 5E1 (the effects of anti-Shh Ab are not illustrated; a, c, e).Anti-ShhN antibody was co-administered to the different conditionedmedia to a final dilution of 1:500. Neuron numbers were assessed and areexpressed as the percentage of TH+ neurons per well, relative tomock-infected treatment in the absence of anti-ShhN antibody(means±SEM); this is illustrated in (g). Compared with the othertreatments, CM ShhN significantly increased the number of TH+ neurons inthe absence but not in the presence of anti-Shh antibody (g).CM-mock+antibody (n=12), 86.68±6.47; CM-mock (n=12), 100.00±6.73;CM-RAd-35+ antibody (n=12), 10162±9.84; CM-RAd-35 (n=12), 97.46±4.12;CM-Shh+antibody (n=12), 112±8.22; CM-Shh (n=12), 159.82±9.51. Two wayANOVA followed by Tukey post hoc analysis indicated that the grouptreated with RAd-ShhN was statistically significantly different(P≦0.001) from all other groups, and there was no statisticallysignificant difference between any of the other treatments. Thisexperiment was repeated at least three times, a-f and g originate fromdifferent experiments. Images were chosen to illustrate neuronalmorphology; quantitation was chosen to determine the statisticalsignificance of the results.

FIG. 4 illustrates the bioreactivity of RAd-ShhN in accordance with anembodiment of the present invention. (a) In vitro bioactivity ofRAd-ShhN, RAd-Gli-1, and RAd-Nurr-1. A schematic view of the mechanismof action of ShhN, and Gli-1, is shown. This illustrates the interactionof ShhN with Ptc and Smo, the release of the inhibition on Smo, and theeventual stimulation of activated Gli-1 translocation into the nucleusto activate further downstream target genes. (b) The bioactivity ofRAd-ShhN and RAd-Gli-1. HeLa cells were infected with RAd-ShhN, orRAd-Gli-1, or a negative control vector, at m.o.i. 200 IU/cell, and 48hours later cells were fixed and the proteins detected with specificprimary antibodies and immunofluorescently labeled secondary antibodies.Most of the ShhN immunoreactivity highlighted the Golgi apparatus,compatible with the intracellular distribution of a secretory protein,while Gli-1 showed both a cytoplasmic and a nuclear localization, asexpected from a transcription factor that has been shown to shuttlebetween the cytoplasm and the nucleus. Differentiation of thepluripotential cell line C3H10T1/2 into osteoblasts was induced uponinfection with RAd-ShhN, or RAd-Gli-1, following infection at m.o.i.200. Alkaline phosphatase (AP) activity was used as a marker forosteoblast differentiation. Both vectors induced AP activity, and thequantitative analysis of AP+ cells is illustrated in (c). (d, e) Thetranscriptional activation mediated by RAd-Nurr-1 is shown. COS-7 cellswere transfected with the reporter plasmid NBRE-Luciferase (d),containing the binding site for Nurr-1, and infected with RAd-Nurr-1 insense orientation or RAd-Nurr-1 in antisense orientation (RAd-Nurr-1AS),used as negative control. (e) Luciferase activity was measured 48 hoursafter. The transcription factor Nurr1 binds to the canonical NBRE domainand induces expression of luciferase. (f) COS-7 cells were infected withRAd-Nurr-1, or a negative control vector, at m.o.i. 200 IU/cell, and 48hours later cells were fixed and immunostained with antibodiesrecognizing Nurr1.

FIG. 5 illustrates retrograde targeting of nigrostriatal dopaminergicneurons throughout the rostrocaudal extent of the substantia nigra inaccordance with an embodiment of the present invention. A recombinantadenovirus encoding the reporter gene thymidine kinase (RAd-TK, 3.2×10⁷IU) was stereotaxically injected into the rat dorsal striatum (AP+1.0mm, ML+3.2, DV−5.0 mm). Retrograde transport of this vector to thesubstantia nigra pars compacta (SNpc) was verified one week after theinjection, by immunostaining of TK protein using specific anti-HSV TKantibodies. (A-P) The expression of TK throughout the rostrocaudal axisof the SNpc from (A) AP −4.8 to (P) AP −6.30.

FIG. 6 illustrates a method for combined retrograde targeting ofsubstantia nigra dopaminergic neurons with both fluoro-gold andadenoviral vectors in accordance with an embodiment of the presentinvention. The method used to retrograde target RAd and fluoro-gold toanatomically overlapping areas in the substantia nigra is illustrated.The method was exhaustively optimized to label a comparable amount ofnigrostriatal neurons (range 30-50) by each method. Also illustrated isthe degeneration of fluoro-gold+ cells after administration of 6-OHDAand the fact that all neurons retrogradely labeled with fluoro-goldindeed are TH-positive. All neurons retrogradely labeled by RAd werealso TH-positive (not illustrated). (a) A coronal section through thestriatum showing colocalization of the fluorescent tracer fluoro-gold(green) and RAd-expressed transgene (red), following their stereotaxicinjection into the rat brain at the level of the dorsal striatum, usingcoordinates identical to those used for the neurotoxicity experiments(scale bar, 1 mm). For these experiments RAd-TK (encoding anintracellular gene product) was injected into the striatum, as describedfor FIG. 5. One week later, the progressive degeneration of thenigrostriatal pathway was induced by injection of 6-OHDA at the samecoordinates used for fluoro-gold and RAd. (b) A coronal section of thesubstantia nigra. Fluoro-gold has been retrogradely transported to thesubstantia nigra (green), and RAd-expressed transgene is detected byimmunocytochemistry (red). Double labeling demonstrates colocalization(yellow) of fluoro-gold (green) and RAd-encoded TK (red) in the neuronsof the SNpc. Expression of the encoded marker transgene was detected byindirect immunofluorescence. Sections illustrated in (c-g) show coronalsections of the substantia nigra immunoreacted for TH and evaluated forthe presence of fluoro-gold. Dopaminergic neurons were detected byimmunofluorescence using a specific anti-TH antibody. Notice thedegeneration of fluoro-gold+neurons after intrastriatal injection of 16μg of 6-OHDA (e.g., e, low-power view of the substantia nigra; g,high-magnification view). In the contralateral side, vehicle (saline)injection in the striatum does not induce degeneration (c and f, c,low-power view of the substantia nigra; f, high-magnification view).Colocalization (yellow) indicates that every fluoro-gold+neuron (green)is TH+(red).

FIG. 7 illustrates effects of gene transfer on substantia nigradopaminergic neurons in accordance with an embodiment of the presentinvention. Adenovirus-mediated gene transfer of 1×10⁸ IU of (a, b)RAd-35, (c, d) RAd-GDNF, (e, f) RAd-ShhN, (g, h) RAd-Gli-1, orRAd-Nurr-1 (not illustrated) was tested against 6-OHDA-inducedneurodegeneration of nigrostriatal cells retrogradely labeled withfluoro-gold. The side injected with 6-OHDA is shown on the left, and thecontrol side is shown on the right. Injection of RAd-GDNF, RAd-ShhN, andRAd-Gli-1 protected a significant amount of nigrostriatal neuronscompared to animals injected with the negative control vector RAd-35.Note the survival of large fluoro-gold-+ neurons in the ipsilateral siteof animals injected with RAd-ShhN (e), (g), and RAd-GDNF (c) comparedwith RAd-35 (a). The quantitative analysis is shown in (i) and alsoindicates the analysis of the animals injected with RAd-Nurr-1. Survivalof nigrostriatal neurons was expressed as a percentage of unlesionedcontralateral neurons. (j) The area occupied by dopamine neurons' cellbodies protected from degeneration after treatment with 1×10⁸ IU ofRAd-ShhN, RAd-Gli-1, or RAd-GDNF was quantified and expressed as apercentage of the neuron soma area in the contralateral site. RAd-GDNF,RAd-ShhN, and RAd-Gli-1 all protected cell body size compared withRAd-35. RAd-GDNF showed the strongest effect. Cell body protection byShhN and Gli-1 was statistically significantly different from that ofanimals injected with RAd-35. The treatment groups were compared byrepeated-measures ANOVA with post hoc Tukey or Dunnet multiplecomparison test; *P≦0.05; ***P≦0.005.

FIG. 8 illustrates effects of gene transfer on striatal dopaminergicinnervation in accordance with an embodiment of the present invention.The column on the left illustrates sections throughout the striatum oftreated animals immunoreacted with an antibody raised against TH, toreveal the density of TH-immunopositive fibers in the striatum. Thelesioned side is on the left, the control side is on the right. Noticethat only animals injected with RAd-GDNF showed a protection of thestriatal dopaminergic fibers. The graph shows the densitometric analysisof TH+ fiber density in the striatum of rats treated with 1×10⁸ IU ofRAd-Shh, RAd-Gli-1, RAd-GDNF, or RAd-35. Degeneration of axonalterminals in the striatum after administration of 6-OHDA is notprevented following the injection of RAd-ShhN, RAd-Gli-1, or RAd-Nurr-1(not shown). Only RAd-GDNF is able to protect TH+ fibers significantly.The treatment groups were compared by repeated-measures ANOVA with posthoc Tukey or Dunnet multiple comparison test; *P≦0.05. Scale bar, 1 mm.

FIG. 9 illustrates transgene expression in the substantia nigra inaccordance with an embodiment of the present invention. RAd-ShhN orRAd-Gli-1 was injected into the striatum and 1 or 4 weeks later animalswere perfusion-fixed and brains were sectioned and probed with specificantibodies for either ShhN or Gli-1. Two rostrocaudal levels are shownfor each condition. There was no specific immunostaining in either thecontralateral substantia nigra or the uninjected animals. Scale bar, 200μm.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); March, Advanced Organic Chemistry Reactions, Mechanisms andStructure 4th ed, J. Wiley & Sons (New York, N.Y. 1992); and Sambrookand Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide oneskilled in the art with a general guide to many of the terms used in thepresent application. One skilled in the art will recognize many methodsand materials similar or equivalent to those described herein, whichcould be used in the practice of the present invention. Indeed, thepresent invention is in no way limited to the methods and materialsdescribed. For purposes of the present invention, the following termsare defined below.

“Beneficial results” include, but are in no way limited to, lessening oralleviating the severity of Parkinson's disease (PD) or itscomplications, preventing or inhibiting it from manifesting, preventingor inhibiting it from recurring, merely preventing or inhibiting it fromworsening, curing PD, reversing the progression of PD, prolonging apatient's life or life expectancy, ameliorating PD, or a therapeuticeffort to effect any of the aforementioned, even if such therapeuticeffort is ultimately unsuccessful.

“Curing” PD includes altering the physiology of the central nervoussystem (“CNS”) and/or its biological components to the point that thedisease cannot be detected after treatment.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting foreign DNA into host cells. Such methods can resultin transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene transfer provides a unique approachfor the treatment of acquired and inherited diseases. A number ofsystems have been developed for gene transfer into mammalian cells. See,e.g., U.S. Pat. No. 5,399,346. Examples of well known vehicles for genetransfer include adenovirus and recombinant adenovirus (RAv),adeno-associated virus (AAV), herpes simplex virus type 1 (HSV-1), andlentivirus (LV).

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“Therapeutically effective amount” as used herein refers to that amountwhich is capable of achieving beneficial results in a patient with PD. Atherapeutically effective amount can be determined on an individualbasis and will be based, at least in part, on consideration of thephysiological characteristics of the mammal, the type of delivery systemor therapeutic technique used and the time of administration relative tothe progression of the disease.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent, slow down and/or lessen the disease even if the treatmentis ultimately unsuccessful.

“AAV vector” refers to any vector derived from an adeno-associated virusserotype, including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-typegenes deleted in whole or in part, preferably the Rep and/or Cap genes,but retain functional flanking inverted terminal repeat (“ITR”)sequences. Functional ITR sequences are generally necessary for therescue, replication and packaging of the AAV virion. Thus, an AAV vectoris defined herein to include at least those sequences required in cisfor replication and packaging (e.g., functional ITRs) of the virus. TheITRs need not be the wild-type nucleotide sequences, and may be altered(e.g., by the insertion, deletion or substitution of nucleotides) solong as the sequences provide for functional rescue, replication andpackaging. A number of adenovirus-based gene delivery systems have alsobeen developed. Human adenoviruses are double-stranded DNA viruses whichenter cells by receptor-mediated endocytosis. These viruses areparticularly well suited for gene transfer because they are easy to growand manipulate and they exhibit a broad host range both in vivo and invitro. Adenovirus is easily produced at high titers and is stable sothat it can be purified and stored. Even in the replication-competentform, adenoviruses generally cause only low level morbidity and aregenerally not associated with human malignancies. For descriptions ofvarious adenovirus-based gene delivery systems, see, e.g., Haj-Ahmad andGraham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol.67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729;Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al.(1993) Human Gene Therapy 4:461-476. The construction of recombinantadeno-associated virus (“rAAV”) vectors has also been described. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PatentPublication Numbers WO 92/01070 (published Jan. 23, 1992) and WO93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell.Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring HarborLaboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology3:533-539; Muzyczka, N. (1992) Current Topics in Microbial. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.

“Recombinant virus” refers to a virus that has been genetically altered(e.g., by the addition or insertion of a heterologous nucleic acidconstruct into the particle).

“AAV virion” refers to a complete virus particle, such as a wild-type(“wt”) AAV virus particle (i.e., including a linear, single-stranded AAVnucleic acid genome associated with an AAV capsid protein coat). In thisregard, single-stranded AAV nucleic acid molecules of eithercomplementary sense (i.e., “sense” or “antisense” strands) can bepackaged into any one AAV virion; both strands are equally infectious.

A “recombinant AAV virion” or “rAAV virion” is defined herein as aninfectious, replication-defective virus composed of an AAV proteinshell, encapsidating a heterologous DNA molecule of interest (e.g.,ShhN, Gli-1) which is flanked on both sides by AAV ITRs. A rAAV virionmay be produced in a suitable host cell which has had an AAV vector, AAVhelper functions and accessory functions introduced therein. In thismanner, the host cell is rendered capable of encoding AAV polypeptidesthat are required for packaging the AAV vector (i.e., containing arecombinant nucleotide sequence of interest) into recombinant virionparticles for subsequent gene delivery.

The term “transfection” is used herein to refer to the uptake of foreignDNA by a cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties, such as a plasmid vector and other nucleic acid molecules,into suitable host cells. The term refers to both stable and transientuptake of the genetic material.

The term “transduction” denotes the delivery of a DNA molecule to arecipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant AAV virion.

The term “heterologous,” as it relates to nucleic acid sequences such asgene sequences and control sequences, denotes sequences that are notnormally joined together and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

“DNA” is meant to refer to a polymeric form of deoxyribonucleotides(i.e., adenine, guanine, thymine and cytosine) in double-stranded orsingle-stranded form, either relaxed or supercoiled. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includessingle- and double-stranded DNA found, inter alia, in linear DNAmolecules (e.g., restriction fragments), viruses, plasmids, andchromosomes. In discussing the structure of particular DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having the sequencehomologous to the mRNA). The term captures molecules that include thefour bases adenine, guanine, thymine and cytosine, as well as moleculesthat include base analogues which are known in the art.

A “gene” or “coding sequence” or a sequence which “encodes” a particularprotein is a nucleic acid molecule that is transcribed (in the case ofDNA) and translated (in the case of mRNA) into a polypeptide in vitro orin viva when placed under the control of appropriate regulatorysequences. The boundaries of the gene are determined by a start codon atthe 5′ (i.e., amino) terminus and a translation stop codon at the 3′(i.e., carboxy) terminus. A gene can include, but is not limited to,cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thegene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always bepresent, so long as the selected coding sequence is capable of beingreplicated, transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region including a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” or “5′,” or “3′” relative toanother sequence, it is to be understood that it is the position of thesequences in the non-transcribed strand of a DNA molecule that is beingreferred to as is conventional in the art.

“Homology” as used herein refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown in the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions which form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. Two DNA or two polypeptide sequences are“substantially homologous” to each other when at least about 80%,preferably at least about 90%, and most preferably at least about 95% ofthe nucleotides or amino acids, respectively, match over a definedlength of the molecules, as determined using the methods above.

“Isolated” as used herein when referring to a nucleotide sequence,refers to the fact that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.Thus, an “isolated nucleic acid molecule which encodes a particularpolypeptide” refers to a nucleic acid molecule that is substantiallyfree of other nucleic acid molecules that do not encode the subjectpolypeptide. However, the molecule may include some additional bases ormoieties that do not deleteriously affect the basic characteristics ofthe composition.

The invention is based on the inventors' study of the activity of thedopaminergic neuron differentiation factor sonic hedgehog (Shh), itsdownstream transcription factor target Gli-1, and an orphan nuclearreceptor, Nurr-1, necessary for the induction of the dopaminergicphenotype of nigrostriatal neurons, in an in vivo model of nigrostriatalneurodegeneration. Experiments demonstrated that all three constructsexpressed the proper molecules and that these had the predictedbiological activities in vitro. The inventors expressed the N-terminalof sonic hedgehog (ShhN) and the Gli-1 and Nurr-1 entire coding regionsfrom highly purified, and quality controlled, replication-defectiveadenoviral vectors injected into the brains of rats and used thedopaminergic growth factor GDNF as a positive control. The neurotoxin6-hydroxydopamine was used to lesion the nigrostriatal dopaminergicinnervation. RAd-ShhN and RAd-Gli-1 protected dopaminergic neuronal cellbodies in the substantia nigra, but not axonal terminals in thestriatum, from 6-OHDA-induced cell death, while RAd-Nurr-1 wasineffective in protecting either cell bodies or axons. RAd-GDNF was ableto protect both the dopaminergic cell bodies and the striatal axonterminals. The inventors' results establish that gene transfer of ShhNand one of its target transcription factors can selectively protectdopaminergic nigrostriatal neuronal cell bodies from a specificneurotoxic insult. Selective protection of nigrostriatal dopaminergiccell bodies by the differentiation factor ShhN and the transcriptionfactor Gli-1 was achieved in a neurotoxic model that eliminates morethan 70% of the nigral neurons under consideration. Differentiation andtranscription factors can thus be used for the treatment ofneurodegeneration by gene therapy.

Shh, secreted by the floor plate, ventralizes the developing neural tubeand induces differentiation of midbrain nigrostriatal dopamine neurons[M. Hynes et al., Induction of midbrain dopaminergic neurons by Sonichedgehog, Neuron, 15:35-44 (1995)]. Shh interacts with its receptorpatched (ptc) and smoothened (smo) [D. Kalderon, Transducing thehedgehog signal. Cell, 103:371-374 (2000)], leading to thephosphorylation and nuclear translocation of the transcription factorGli-1 [I. A. A. Ruiz et al., Hedgehog-Gli signalling and the growth ofthe brain, Nat. Rev. Neurosci., 3:24-33 (2002); R. J. Hardy,Dorsoventral patterning and oligodendroglial specification in thedeveloping central nervous system, J. Neurosci. Res., 50:139-145 (1997)]and activation of downstream genes [C. C. Hui et al., Expression ofthree mouse homologs of the Drosophila segment polarity gene cubitusinterruptus, Gli, Gli-2, and Gli-3, in ectoderm-and mesoderm-derivedtissues suggests multiple roles during postimplantation development,Dev. Biol., 162:402-413 (1994); J. Lee et al., Gli1 is a target of Sonichedgehog that induces ventral neural tube development, Development,124:2537-2552 (1997); K. A. Platt et al., Expression of the mouse Gliand Pk genes is adjacent to embryonic sources of hedgehog signalssuggesting a conservation of pathways between flies and mice, Mech.Dev., 62:121-135 (1997); H. Sasaki et al., A binding site for Gliproteins is essential for HNF-3beta floor plate enhancer activity intransgenics and can respond to Shh in vitro, Development, 124:1313-1322(1997)].

ShhN protects cultures of fetal dopamine neurons from MPP+ toxicity [N.Miao et al., Sonic hedgehog promotes the survival of specific CNS neuronpopulations and protects these cells from toxic insult in vitro, J.Neurosci., 17:5891-5899 (1997)] and regulates the differentiation andproliferation of neuronal stem cells [K. Lai et at, Sonic hedgehogregulates adult neural progenitor proliferation in vitro and in vivo,Nat. Neurosci., 6:21-27 (2003); N. Matsuura et al., Sonic hedgehogfacilitates dopamine differentiation in the presence of a mesencephalicglial cell line, J. Neurosci., 21:4326-4335 (2001); T. E. Kim et al.,Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes inthe Nurr1-overexpressing neural stem cell. Biochem. Biophys. Res.Commun., 305:1040-1048 (2003)]. Further, Shh peptide injected directlyinto the brains of rodents and marmosets has beneficial effects inexperimental models of PD [E. Bezard et al., Sonic hedgehog is aneuromodulator in the adult subthalamic nucleus, FASEB J., 17:2337-2338(2003); B. Dass et al., Behavioural and immunohistochemical changesfollowing supranigral administration of sonic hedgehog in1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets,Neuroscience, 114:99-109 (2002); K. Tsuboi et at, Intrastriatalinjection of sonic hedgehog reduces behavioral impairment in a rat modelof Parkinson's disease, Exp. Neurol., 173:95-104 (2002)]. Nurr-1 is anorphan nuclear receptor necessary for the expression of the dopaminergicphenotype of developing nigrostriatal neurons, such as tyrosinehydroxylase, dopamine transporter [R. H. Zetterstrom et al, Dopamineneuron agenesis in Nurr1-deficient mice, Science, 276:248-250 (1997)].Shh, ptc, smo, Gli-1, and Nurr-1 are present in the adult rodent brain[D. Charytoniuk et al., Sonic Hedgehog signalling in the developing andadult brain, J. Physiol. Paris, 96:9-16 (2002); E. Traiffort et al.Discrete localizations of hedgehog signalling components in thedeveloping and adult rat nervous system, Eur. J. Neurosci., 11:3199-3214(1999); E. Traiffort et al., Regional distribution of Sonic Hedgehog,patched, and smoothened mRNA in the adult rat brain, J. Neurochem.,70:1327-1330 (1998)].

To test the hypothesis that Shh, Gli-1, or Nurr-1 protects dopaminenigrostriatal neurons from neurotoxin-induced neurodegeneration theinventors constructed RAd vectors expressing ShhN (RAd-ShhN), Gli-1(RAd-Gli-1), or Nurr-1 (RAd-Nurr-1) under the control of the majorimmediate early human cytomegalovirus promoter (hCMV) and compared theseto GDNF (RAd-GDNF) and a control vector expressing β-galactosidase(RAd-35). RAd-ShhN and RAd-Gli-1 protected nigrostriatal dopaminergiccell bodies, but not their striatal terminals, from 6-OHDA-inducedneurodegeneration, while RAd-Nurr-1 was ineffective. These resultsindicate that nigrostriatal dopaminergic cell bodies can be protectedfrom neurotoxin-induced cell death independent of the maintenance oftheir axonal terminals. While not wishing to be bound by any particulartheory, it is believed that ShhN and Gli-1 may be neuroprotectivethrough the activation of mechanisms different from those of GDNF, whichprotects both cell bodies and striatal terminals.

In further experimental procedures, the inventors found thatneuron-specific expression of Gli-1 using the neuron-specific Tα1α-tubulin promoter was neuroprotective and its efficiency was comparableto the pancellular strong viral hCMV promoter [D. Suwelack et al.,Neuronal expression of the transcription factor Gli1 using the Tα1α-tubilin promoter is neuroprotective in an experimental model ofParkinson's disease, Gene Therapy, 11:1742-1752 (2004)].

The invention includes compositions and methods for the treatment of PDusing ShhN and/or Gli-1, either through gene therapeutic approaches ordirect peptide injection. More specifically, the invention includesmethods of treating PD by administering a therapeutically effectiveamount of ShhN and/or Gli-1 to a mammal. In one embodiment of thepresent invention, the mammal is a human. The ShhN and/or Gli-1 may beformulated into an appropriate pharmaceutical composition for use inconnection with the gene therapeutic and/or direct peptide deliverytechniques as contemplated by alternate embodiments of the presentinvention.

The inventive therapeutics may be administered by any appropriatetechnique, as will be readily appreciated by those of skill in the art.With respect to embodiments of the present invention that incorporateShhN and/or Gli-1 therapeutics, the therapy may be administered by agene therapeutic approach. For instance, rAAV virions includingheterologous DNA corresponding to a ShhN and/or Gli-1 coding sequencemay be generated by any conventional technique known in the art. By wayof example, the recombinant AAV virions of the present invention,including the ShhN or Gli-1 DNA of interest, can be produced by astandard methodology that generally involves the steps of: (1)introducing an AAV vector into a host cell; (2) introducing an AAVhelper construct into the host cell, where the helper construct includesAAV coding regions capable of being expressed in the host cell tocomplement AAV helper functions missing from the AAV vector; (3)introducing one or more helper viruses and/or accessory function vectorsinto the host cell, wherein the helper virus and/or accessory functionvectors provide accessory functions capable of supporting efficient rAAVvirion production in the host cell; and (4) culturing the host cell toproduce rAAV virions. The AAV vector, AAV helper construct and thehelper virus or accessory function vector(s) can be introduced into thehost cell either simultaneously or serially, using standard transfectiontechniques. Examples of such techniques are described in greater detailin the ensuing Examples herein. In accordance with various embodimentsof the present invention, it may be particularly beneficial to constructthe viral vectors of the instant invention such that ShhN and/or Gli-1are under the control of, for example, the hCMV promoter or the Tα1promoter.

AAV vectors are constructed using known techniques to at least provide,as operatively linked components in the direction of transcription, (a)control elements including a transcriptional initiation region, (b) theShhN and/or Gli-1 DNA of interest and (c) a transcriptional terminationregion. Moreover, any coding sequence sufficiently homologous to theShhN and/or Gli-1 coding sequence so as to exhibit functional propertiessubstantially similar to the ShhN and/or Gli-1 coding sequence may beused in connection with alternate embodiments of the present invention.The control elements are selected to be functional in the targetedcell(s). The resulting construct, which contains the operatively linkedcomponents, may be bounded (5′ and 3′) with functional AAV ITRsequences. The nucleotide sequences of AAV ITR regions are known. See,e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I.“Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence.AAV ITRs used in the vectors of the invention need not have a wild-typenucleotide sequence, and may be altered (e.g., by the insertion,deletion or substitution of nucleotides). Additionally, AAV ITRs may bederived from any of several AAV serotypes, including, withoutlimitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, and the like.Furthermore, 5′ and 3′ ITRs that flank a selected nucleotide sequence inan AAV expression vector need not necessarily be identical or derivedfrom the same AAV serotype or isolate, so long as they function asintended (i.e., to allow for excision and replication of the boundedShhN and/or Gli-1 nucleotide sequence of interest).

Therefore, in accordance with an embodiment of the invention, the rAAVvirions including a ShhN and/or Gli-1 coding sequence are delivered to amammal in a sufficient quantity and by a sufficient delivery route so asto effect gene transfer. This may provide an effective treatment for PDin the mammal. In an alternate embodiment of the present invention, thismay protect dopaminergic nigrostriatal neuronal cell bodies from6-OHDA-induced neurotoxicity.

In an alternate embodiment of the present invention, a quantity of ShhNand/or Gli-1 peptide may be directly administered to a mammal in atherapeutically effective amount to treat PD and/or to protectdopaminergic nigrostriatal neuronal cell bodies from 6-OHDA-inducedneurotoxicity.

In various embodiments, the present invention provides pharmaceuticalcompositions (in connection with gene therapeutics and direct peptideadministration techniques) including a pharmaceutically acceptableexcipient along with either a therapeutically effective amount of aviral vector for delivery of ShhN and/or Gli-1 or a therapeuticallyeffective amount of ShhN and/or Gli-1 protein. “Pharmaceuticallyacceptable excipient” means an excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic, anddesirable, and includes excipients that are acceptable for veterinaryuse as well as for human pharmaceutical use. Such excipients may besolid, liquid, semisolid, or, in the case of an aerosol composition,gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders. In one embodiment of thepresent invention the inventive compositions are injected directly intothe brain of a mammal.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

The present invention is also directed to a kit for the treatment of PD.The kit is useful for practicing the inventive method of treating PD.The kit is an assemblage of materials or components, including at leastone of the inventive compositions. Thus, in some embodiments the kitcontains a composition including a viral vector expressing ShhN and/oror, in an alternate embodiment, the kit contains a composition includingShhN and/or Gli-1 peptides, as described above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments of thekit are configured for the purpose of treating cultured mammalian cells.Other embodiments are configured for the purpose of treating mammaliancells in viva (i.e., for treating mammalian subjects in need oftreatment, for example, subjects with PD). In one embodiment, the kit isconfigured particularly for the purpose of treating human subjects.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as the treatment of PD. Optionally, the kit also contains otheruseful components, such as, diluents, buffers, pharmaceuticallyacceptable carriers, specimen containers, syringes, stents, catheters,pipetting or measuring tools, or other useful paraphernalia as will bereadily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in polynucleotide-based or peptide-based systems. As usedherein, the term “package” refers to a suitable solid matrix or materialsuch as glass, plastic, paper, foil, and the like, capable of holdingthe individual kit components. Thus, for example, a package can be aglass vial used to contain suitable quantities of an inventivecomposition containing nucleic acid or peptide components. The packagingmaterial generally has an external label which indicates the contentsand/or purpose of the kit and/or its components.

In another embodiment, the present invention includes various in vivomodels of nigrostriatal neurodegeneration, using a non-human mammal thatcarries in at least a portion of the cells of its brain at least oneexogenous ShhN or Gli-1 DNA. Such animal models may be useful for avariety of purposes, including studying a number of diseases andphysiologic conditions (e.g., those described herein), as well asscreening therapeutic candidates for the treatment of such diseases andphysiologic conditions, and still further uses that will be readilyapparent to those of skill in the art.

The inventors' experiments demonstrate that the differentiation factorShhN can act as a trophic factor for embryonic dopaminergic neurons inprimary cultures, and, similar to its downstream transcriptionalactivator, Gli-1, they both protect a significant percentage of adultdopaminergic nigrostriatal neuronal cell bodies from 6-OHDA-inducedneurotoxicity. ShhN and Gli-1 protected only the nigral dopaminergiccell bodies from neurodegeneration and not the striatal axonal terminalsof these neurons. The positive control vector used expressing GDNF, onthe other hand, protected both the nigral cell bodies and thedopaminergic terminals. It is important to note that GDNF protected ahigher percentage of nigral cell bodies (≈80%) compared to thepercentage of striatal dopaminergic innervation (≈60%). The inventors'negative control vector expressing the reporter β-galactosidase and theRAd-Nurr-1 vector, however, were unable to protect either the cellbodies or the dopaminergic axon terminals in the striatum. The use ofinternal positive and negative control vectors provided stringentmechanisms to identify both active and inactive factors able to protectnigral cell bodies from neurotoxic degeneration.

6-OHDA reduced the size of dopaminergic neuronal cell bodies in thesubstantia nigra. RAd-GDNF, which showed the greatest capacity tomaintain the axonal terminals of nigrostriatal neurons, also achievedthe greatest protection of cell body size in the substantia nigra. WhileShhN and Gli-1 were less potent, they significantly protected againstthe neurotoxin-induced soma atrophy. Thus, while GDNF was more effectivein preserving nigral dopamine neuron numbers, striatal tyrosinehydroxylase axon terminal density, and nigral dopamine soma size, ShhNand Gli-1 had a protective effect on neuronal numbers and nigral somaarea, but were completely ineffective in protecting the striataldopaminergic innervation from neurotoxin-induced degeneration. Thus,ShhN and Gli-1 protected partially against the denervation-induced somaatrophy, without preserving the density of dopaminergic striatalinnervation.

Administration of GDNF into the substantia nigra has been previouslybeen shown to protect mainly the nigral dopaminergic cell bodies [A.Bjorklund et al., Towards a neuroprotective gene therapy for Parkinson'sdisease: use of adenovirus, AAV and lentivirus vectors for gene transferof GDNF to the nigrostriatal system in the rat Parkinson model, BrainRes., 886:82-98 (2000)] but not the striatal axonal terminals. Deliveryof GDNF to the striatum has been shown to provide neuroprotection toboth nigral cell bodies and axonal terminals, as seen in the experimentsperformed and described herein and those described by others [D. L.Choi-Lundberg et al., Dopaminergic neurons protected from degenerationby GDNF gene therapy, Science, 275:838-841 (1997); A. Bjorklund et al.,Towards a neuroprotective gene therapy, for Parkinson's disease: use ofadenovirus, AAV and lentivirus vectors for gene transfer of GDNF to thenigrostriatal system in the rat Parkinson model, Brain Res., 886:82-98(2000); D. L. Choi-Lundberg et al., Behavioral and cellular protectionof rat dopaminergic neurons by an adenoviral vector encoding glial cellline-derived neurotrophic factor, Exp. Neural., 154:261-275 (1998); B.Connor et al., Differential effects of glial cell line-derivedneurotrophic factor (GDNF) in the striatum and substantia nigra of theaged Parkinsonian rat, Gene Ther., 6:1936-1951 (1999); B. Connor et al.,Glia1 cell line-derived neurotrophic factor (GDNF) gene deliveryprotects dopaminergic terminals from degeneration, Exp. Neurol,169:83-95 (2001)]. Further, the anti-apoptotic protein XIAP has alsobeen shown to protect predominantly nigral cell bodies upon deliveryusing recombinant adenovirus into the striatum of mice [O. Eberhardt etal., Protection by synergistic effects of adenovirus-mediatedX-chromosome-linked inhibitor of apoptosis and glial cell line-derivedneurotrophic factor gene transfer in the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson'sdisease, J. Neurosci., 20:9126-9134 (2000)]. Thus, there is precedencefor the selective neuroprotection of nigral dopaminergic cell bodies,even when using experimental paradigms comparable to those describedherein.

The increased effectiveness of GDNF may be related to its capacity topreserve the striatal axon terminals [B. Connor et al., Differentialeffects of glial cell line-derived neurotrophic factor (GDNF) in thestriatum and substantia nigra of the aged Parkinsonian rat, Gene Ther.,6:1936-1951 (1999); A. Bilang-Bleuel et al., Intrastriatal injection ofan adenoviral vector expressing glial-cell-line-derived neurotrophicfactor prevents dopaminergic neuron degeneration and behavioralimpairment in a rat model of Parkinson disease, Proc. Natl. Acad. Sci.USA, 94:8818-8823 (1997)]. Although recently untoward effects of GDNFhave been described at long times following GDNF delivery [B.Georgievska et al., Aberrant sprouting and downregulation of tyrosinehydroxylase in lesioned nigrostriatal dopamine neurons induced bylong-lasting overexpression of glial cell line derived neurotrophicfactor in the striatum by lentiviral gene transfer, Exp. Neurol.,177:461-474 (2002); C. Rosenblad et al., Long-term striataloverexpression of GDNF selectively downregulates tyrosine hydroxylase inthe intact nigrostriatal dopamine system, Eur. J. Neurosci., 17:260-270(2003)], the experimental design upon which the present invention isbased was not designed to examine these specifically, and thus, theywere not uncovered. Intrastriatal 6-OHDA causes a slow degeneration ofnigral neurons. Hence, although not wishing to be bound by anyparticular theory, it is believed that nigral neuron survival coulddepend on the continued retrograde transport of the adenoviruses, or thegrowth factors encoded by them, to the substantia nigra. It has beenrecently demonstrated that Shh and its receptor Ptc can also beendocytosed in neural plate explants; suggesting that this event may belinked to the mechanism of Shh signal transduction [J. P. Incardona etal., Receptor-mediated endocytosis of soluble and membrane-tetheredSonic hedgehog by Patched-1, Proc. Natl. Acad. Sci. USA, 97:12044-12049(2000)]. These mechanisms will operate only in the presence of intactaxon terminals, and the loss of striatal axons will thus compromise theeffectiveness of RAd-ShhN and RAd-Gli-1. Retrograde transport of RAdshas been demonstrated by the inventors in this paradigm and has alsobeen described by others [D. L. Choi-Lundberg et al., Behavioral andcellular protection of rat dopaminergic neurons by an adenoviral vectorencoding glial cell line-derived neurotrophic factor, Exp. Neurol.,154:261-275 (1998); V. Ridoux et al., Adenoviral vectors as functionalretrograde neuronal tracers, Brain Res., 648:171-175 (1994)].

Nurr-1, despite being indispensable during early brain development forthe expression of the dopaminergic phenotype [R. H. Zetterstrom et al.,Dopamine neuron agenesis in Nurr1-deficient mice, Science, 276:248-250(1997)], had no effect on the survival of nigral dopamine neurons in theinventors' experimental paradigm. Nurr-1 is a powerful factor thatcontributes to the determination of the dopaminergic phenotype duringneuronal differentiation, most possibly by heterodimerization with theretinoid X receptor [A. Wallen-Mackenzie et al., Nurr1-RXR heterodimersmediate RXR ligand-induced signaling in neuronal cells, Genes Dev.,17:3036-3047 (2003)]. Thus, Nurr-1 activates dopaminergic-specific genesin neuronal stem cells, and allows the differentiation of neuronal stemcells along a dopaminergic pathway, and has also been shown to have aneuroprotective effect in mouse neural stem cells [K. Sakurada et al.,Nurr1, an orphan nuclear receptor, is a transcriptional activator ofendogenous tyrosine hydroxylase in neural progenitor cells derived fromthe adult brain. Development, 126:4017-4026 (1999); T. E. Kim et al.,Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes inthe Nurr1-overexpressing neural stem cell, Biochem. Biophys. Res.Commun., 305:1040-1048 (2003); J. Wagner et al., Induction of a midbraindopaminergic phenotype in Nurr1-overexpressing neural stem cells by type1 astrocytes, Nat. Biotechnol., 17:653-659 (1999); J. Y. Kim et al.,Dopaminergic neuronal differentiation from rat embryonic neuralprecursors by Nurr1 overexpression, J. Neurochem., 85:1443-1454 (2003);J. Satoh et al., The constitutive and inducible expression of Nurr1, akey regulator of dopaminergic neuronal differentiation, in human neuraland non-neural cell lines, Neuropathology, 22:219-232 (2002); M. A. Leeet al., Overexpression of midbrain-specific transcription factor Nurr1modifies susceptibility of mouse neural stem cells to neurotoxins,Neurosci. Lett., 333:74-78 (2002)]. In summary, the role of Nurr-1 inpromoting the development of the dopaminergic phenotype of midbrainneurons has been studied during ontogenesis, but its role in adults hasnot been thoroughly evaluated. That it does play a role can be surmisedby its being essential for the expression of Ret, a central component ofGDNF signaling, in midbrain dopamine neurons [A. A. Wallen et al.,Orphan nuclear receptor Nurr1 is essential for Ret expression inmidbrain dopamine neurons and in the brain stem. Mol. Cell. Neurosci.,18:649-663 (2001)]. Thus, the lack of effect of Nurr-1 could be due tothe high constitutive levels of Nurr-1 already present in the adultbrain, its exclusive cell-autonomous function, or its lack ofneuroprotective action against neurotoxins [R. H. Zetterstrom et al.,Retinoid X receptor heterodimerization and developmental expressiondistinguish the orphan nuclear receptors NGFI-B, Nurr1, and Nor1, Mol.Endocrinol., 10:1656-1666 (1996); R. H. Zetterstrom et al., Cellularexpression of the immediate early transcription factors Nurr1 and NGFI-Bsuggests a gene regulatory role in several brain regions including thenigrostriatal dopamine system, Brain Res. Mol. Brain. Res., 41:111-120(1996)].

Previously, GDNF has been successfully used in several animal models ofPD [M. C. Bohn, A commentary on glial cell line-derived neurotrophicfactor (GDNF): from a glial secreted molecule to gene therapy, Biochem.Pharmacol., 57:135-142 (1999)]. However, in a clinical trial,intraventricular administration of GDNF protein failed to preventnigrostriatal degeneration [J. H. Kordower et al., Clinicopathologicalfindings following intraventricular glial-derived neurotrophic factortreatment in a patient with Parkinson's disease, Ann. Neurol.,46:419-424 (1999)]; it did not improve parkinsonism, and side effectsincluding weight loss, paresthesias, and hyponathremia were reported [J.G. Nutt et al., Randomized, double-blind trial of glial cellline-derived neurotrophic factor (GDNF) in PD, Neurology, 60:69-73(2003)]. In a more recent clinical trial in which GDNF was injecteddirectly into the affected striatum of PD patients, potentiallytherapeutic results were forthcoming [S. S. Gill et al., Direct braininfusion of glial cell line-derived neurotrophic factor in Parkinsondisease, Nat. Med., 9:589-595 (2003)]. This indicates that directdelivery of neuronal growth factors into target brain areas may beeffective in human patients. Methods such as gene therapy, by providingsustained and regulated growth factor, may thus become the deliverymethod of choice for the treatment of PD.

There has been one previous report suggesting that ShhN peptideprotected DA neurons in primary cultures from MPP+-inducedneurodegeneration [N. Miao et al., Sonic hedgehog promotes the survivalof specific CNS neuron populations and protects these cells from toxicinsult in vitro, J. Neurosci., 17:5891-5899 (1997)]. More recently, ShhNpeptide has been proposed to act as a neuromodulator in the adultsubthalamic nucleus [E. Bezard et al., Sonic hedgehog is aneuromodulator in the adult subthalamic nucleus, FASEB J., 17:2337-2338(2003)] and to protect partially from 6-OHDA-induced neurotoxicity inrodents [K. Tsuboi et al., Intrastriatal injection of sonic hedgehogreduces behavioral impairment in a rat model of Parkinson's disease,Exp. Neurol. 173:95-104 (2002)] and marmosets [B. Dass et al.,Behavioural and immunohistochemical changes following supranigraladministration of sonic hedgehog in1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets,Neuroscience, 114:99-109 (2002)]. However, the inventors are the firstto describe that, in vivo, ShhN or the transcription factor downstreamof Shh signaling, Gli-1, expressed by a recombinant viral gene transfervector, protects dopaminergic nigrostriatal neurons fromneurodegeneration. In the inventors' experiments, RAd-GDNF was moreeffective than either RAd-ShhN or RAd-Gli-1, similar to results obtainedwhen ShhN peptide was injected directly into the striatum and comparedto infused GDNF [K. Tsuboi et al., Intrastriatal injection of sonichedgehog reduces behavioral impairment in a rat model of Parkinson'sdisease, Exp. Neurol., 173:95-1.04 (2002)].

In the inventors' experiments, RAd-ShhN or RAd-Gli-1 protected asignificant percentage of dopaminergic nigrostriatal neurons fromneurodegeneration; although behavioral protection could not be detectedwith RAd-GDNF. RAd-ShhN, or RAd-Gli-1, because the inventors could notdetect any rotational asymmetries in response to the lesion. Thus, theexperimental model did not allow for the testing for any behavioralprotection and is thus a model of neuroanatomical neuroprotection (datanot shown). This correlated with the lack of neuroprotection of striataldopaminergic terminals, since behavioral neuroprotection of striatalfunction is contingent on the preservation of the dopaminergic striatalinnervation. However, it was recently reported that ShhN peptideinjected directly into the striatum prevented behavioral modificationsinduced by 6-OHDA and protected some of the dopaminergic innervationdensity, but was less powerful compared to GDNF [K. Tsuboi et al.,Intrastriatal injection of sonic hedgehog reduces behavioral impairmentin a rat model of Parkinson's disease, Exp. Neurol. 173:95-104 (2002)].Different results obtained by both groups could be explained by therepeated and presumably higher doses of ShhN peptide injected directlyinto the striatum [K. Tsuboi et al., Intrastriatal injection of sonichedgehog reduces behavioral impairment in a rat model of Parkinson'sdisease, Exp. Neurol., 173:95-104 (2002)] and/or other experimentaldifferences between both sets of experiments.

Differences in the effectiveness between GDNF and ShhN/Gli-1 could beattributed to differences in the density of receptors and signalingpathways activated by either factor. The receptors for ShhN, Ptc andSmo, have been identified in the adult rat brain in several regions,including striatum and midbrain [E. Traiffort et al., Regionaldistribution of Sonic Hedgehog, patched, and smoothened mRNA in theadult rat brain, J. Neurochem., 70:1327-1330 (1998)]. However, thedensity of these receptors in the adult brain has not been determined.For GDNF, however, it is clear that its receptors are expressed atphysiologically relevant levels in the adult brain of various species,including human [A. Josephson et al., GDNF and NGF family members andreceptors in human fetal and adult spinal cord and dorsal root ganglia,J. Comp. Neurol., 440:204-217 (2001); T. C. Burazin et al., Localizationof GDNF/neurturin receptor (c-ret, GFRalpha-1 and alpha-2) mRNAs inpostnatal rat brain: differential regional and temporal expression inhippocampus, cortex and cerebellum, Brain Res. Mol. Brain. Res.,73:151-171 (1999); J. P. Golden et al., Expression of neurturin, GDNF,and GDNF family-receptor mRNA in the developing and mature mouse, Exp.Neurol., 158:504-528 (1999); J. P. Golden et al., Expression ofneurturin, GDNF, and their receptors in the adult mouse CNS, J. Comp.Neurol., 398:139-150 (1998); G. Paratcha et al., The neural celladhesion molecule NCAM is an alternative signaling receptor for GDNFfamily ligands, Cell, 113:867-879 (2003)].

Although not wishing to be bound by any particular theory, it isbelieved that differences in signaling pathways activated by GDNF orShhN/Gli-1 most likely explain their different activities. GDNF signalsthrough binding to GRF1α and its interactions with the Ret-receptortyrosine kinase. The expression of GRF1α is regulated by TGFβ, and retis under the control of Nurr-1 [A. A. Wallen et al., Orphan nuclearreceptor Nurr1 is essential for Ret expression in midbrain dopamineneurons and in the brain stem, Mol. Cell. Neurosci., 18:649-663 (2001);H. Sariola et al., Novel functions and signalling pathways for GDNF, J.Cell Sci., 116 Pt. 19:3855-3862 (2003); M. S. Airaksinen et al., TheGDNF family: signalling, biological functions and therapeutic value.Nat. Rev. Neurosci., 3:383-394 (2002)]. More recently, it has also beendemonstrated that GDNF can bind to heparan sulfate proteoglycans toactivate the Met-receptor tyrosine kinase or bind to NCAM, leading tothe activation of Fyn and FAK. The rescuing of nigrostriatal neuronsfrom neurotoxin-induced toxicity has not yet been linked to individualsignaling pathways.

Shh signals through its interactions with Ptc and Smo, leading to theactivation of a macromolecular complex consisting of Suppressor offused, Fused, and protein kinase A, which phosphorylates and activatesGli proteins [I. A. A. Ruiz et al., Hedgehog-Gli signalling and thegrowth of the brain. Nat. Rev. Neurosci., 3:24-33 (2002)]. Although theoverall signaling pathways are not completely elucidated in the CNS, theinventors' data suggest that Gli-1 is downstream of Shh signaling.

Thus, the inventors have demonstrated that a single intrastriatalinjection of a RAd encoding ShhN or its downstream transcriptionalactivator Gli-1 protects dopamine neurons against 6-OHDA neurotoxicityin vivo and also protected these cells from neurotoxin-induced cell bodyatrophy. Nevertheless, this treatment did not prevent the dopaminergicdenervation of the striatum. This demonstrates the feasibility of usingtranscriptional gene therapy to mimic neuroprotective signals to bypassany limited availability of receptors or signaling cascades ofneurotrophic factors. This strategy may be used both in the context oftherapeutic applications and to determine which signaling pathwaymediates particular effects of a given neurotrophic factor. Theexperimental results suggest that adenovirus-mediated gene transferusing Shh or downstream elements of its signaling pathways represents anew strategy to prevent progressive degeneration of dopamine-containingneurons in the substantia nigra in disorders such as PD.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention

Example 1 Preparation of Cell Lines

Human embryonic kidney cells (293 transformed with E1 from Ad5) wereobtained from Microbix, Biosystems Inc. (Toronto, Ontario; Canada); HeLaand BHK cells were purchased form the European Collection of Animal CellCultures (Porton Down. Salisbury; UK). These cells were grown usingcomplete minimal essential medium Eagle (MEM; fetal bovine serum 10%,penicillin/streptomycin 1%, L-glutamine 1%, MEM nonessential aminoacids) and incubated at 37° C. with 5% CO₂ [P. Lowenstein et al.Protocols for Gene Transfer in Neuroscience: Towards Gene Therapy ofNeurological Disorders, Wiley, Chichester (1996)]. C3H/10T1/2 cells, amouse embryo mesenchymal cell line that can be differentiated intocartilage and bone, were used to test the bioactivity of ShhN and Gli-1.Differentiation induces alkaline phosphatase expression detectedhistochemically. C3H/10T1/2 cells were grown in basal Eagle medium withEarle's BSS 90%, supplemented with 10% heat-inactivated fetal bovineserum in 25-cm² flasks and incubated at 37° C. with 5% CO₂ and passagedat 60% confluency.

Example 2 Preparation of Ventral-Mesencephalic Primary Cultures

Pregnant Sprague-Dawley rats were killed by neck dislocation on dayE14.5 [K. Shimoda et al., A high percentage yield of tyrosinehydroxylase-positive cells from rat E14 mesencephalic cell culture,Brain Res., 586:319-331 (1992)]. The uterus was removed and transferredto ice-cold buffer, where fetuses were removed until dissection under astereomicroscope as described in detail elsewhere [K. Shimoda et al., Ahigh percentage yield of tyrosine hydroxylase-positive cells from ratE14 mesencephalic cell culture, Brain Res., 586:319-331 (1992); A. F.Shering et al., Cell type-specific expression in brain cell culturesfrom a short human cytomegalovirus major immediate early promoterdepends on whether it is inserted into herpesvirus or adenovirusvectors. J. Gen. Viral., 78:445-459 (1997)]. Neocortical cultures wereprepared as described earlier [A. F. Shering et al., Cell type-specificexpression in brain cell cultures from a short human cytomegalovirusmajor immediate early promoter depends on whether it is inserted intoherpesvirus or adenovirus vectors, J. Gen. Virol., 78:445-459 (1997)],and midbrain cultures were prepared as in Shimoda et al. [K. Shimoda etal., A high percentage yield of tyrosine hydroxylase-positive cells fromrat E14 mesencephalic cell culture, Brain Res., 586:319-331 (1992)].Glia1 cells from the neocortical cultures were immunostained withantibodies against glial fibrillary acidic protein, andventral-mesencephalic (VM) midbrain cultures were shown to be enrichedin dopaminergic neurons by immunostaining with antibodies to tyrosinehydroxylase.

Serum-containing or serum-free media were prepared as follows.Serum-free medium consisted of Dulbecco's modified Eagles medium(DMEM)-F12 (1:1; obtained from Life Technologies) containing 2 mML-glutamine (obtained from Life Technologies), 100 units ofpenicillin/ml, 100 units of streptornycin/ml (obtained from LifeTechnologies), 33 mM glucose (obtained from Sigma), and the N1supplements [5 μg/ml insulin (obtained from Sigma), 5 μg/ml transferrin(obtained from Sigma), 2×10⁻⁸ M progesterone (obtained from Sigma), 100μM putrescine (obtained from Sigma), and 3×10⁻⁸ M selenium (as Na₂SeO₃)(obtained from Sigma)]. Serum containing medium was DMEM-F12 (1:1;obtained from Life Technologies) containing 10% fetal bovine serum(obtained from Life Technologies), 4.0 mM glutamine (obtained from LifeTechnologies), 100 units of penicillin/ml, and 100 units ofstreptomycin/ml (obtained from Life Technologies). To prepareconditioned media (CM) from either BHK cells or glial cultures, cellswere infected for 6 hours with an m.o.i. of 300 for each virus and thenincubated for a further 48 hours. At this time medium was removed,centrifuged, and filtered with a 0.2-μm Sartorius filter. Conditionedmedium was diluted 1:1 in fresh medium to maintain cell viability.

Example 3 Construction of Recombinant Adenovirus

The cDNA encoding Shh amino-terminal gene product was excised byenzymatic digestion with EcoRI/HindIII from a recombinant pBluescript IIplasmid provided by Dr. P. Beachy, John Hopkins University (Baltimore,Md.). The full-length (3.6 kb) HindIII/Xbal insert of human (Homosapiens) Gli-1 cDNA clone pGL12 was kindly provided by Dr. BertVolgestein, Johns Hopkins University, and the full-length (0.7 kb)BamHI/XhoI insert of rat GDNF cDNA clone pCDNA-GDNF was made availableby Dr. Ira Black (UMDNJ, NJ). These inserts were then cloned into theshuttle vector pAL119, yielding pALShhN, pALGIi-1, and pALGDNFcontaining the ShhN, the Gli-1, or the GDNF coding region, respectively,in the sense orientation with respect to the major immediate early hCMVpromoter of pAL 119 [A. F. Shering et al., Cell type-specific expressionin brain cell cultures from a short human cytomegalovirus majorimmediate early promoter depends on whether it is inserted intoherpesvirus or adenovirus vectors, J. Gen. Virol., 78:445-459 (1997)].The orientation of the cloned transgenes was determined by restrictionanalysis with EcoRI or HindIII for ShhN and HindIII or SacI for Gli-1[M. M. Hitt et al. Human adenovirus vectors for gene transfer intomammalian cells, Adv. Pharmacol., 40:137-206 (1997)]. Recombinantadenoviruses RAd-ShhN, RAd-Gli-1, and RAd-GDNF expressing ShhN, Gli-1,and GDNF, respectively, under the control of the major immediate earlyhCMV promoter were generated by cotransfection of the shuttle plasmidpALShhN, pALGli-1, or pALGDNF with the Ad5 genomic plasmid pJM17 into293 HEK cells as earlier described [P. Lowenstein et al., Protocols forGene Transfer in Neuroscience: Towards Gene Therapy of NeurologicalDisorders, Wiley, Chichester (1996); A. F. Shering et al., Celltype-specific expression in brain cell cultures from a short humancytomegalovirus major immediate early promoter depends on whether it isinserted into herpesvirus or adenovirus vectors, J. Gen. Virol.,78:445-459 (1997); T. Southgate et al., Gene transfer into neural cellsin vivo using adenoviral vectors, In: C. R. Gerfen, R. McKay, M. A.Rogawski, D. R. Sibley and P. Skolnick, Editors, Current Protocols inNeuroscience, Wiley, New York (2000), 4.23.1-4.23.40]. After molecularcharacterization of the RAds, they were purified by three rounds ofdilution limiting assay, scaled up, and purified by CsCl₂ gradient, andthe presence and identity of the transgenes were confirmed byrestriction and Southern blot analysis.

The construction and use of RAd-35 (expressing β-galactosidase) andRAd-TK (expressing HSV-1 TK), both under the control of the hCMVpromoter, were described earlier [R. A. Dewey et at, Chronic braininflammation and persistent herpes simplex virus 1 thymidine kinaseexpression in survivors of syngeneic glioma treated byadenovirus-mediated gene therapy: implications for clinical trials, Nat.Med., 5:1256-1263 (1999); A. J. Zermansky et al., Towards global andlong-term neurological gene therapy: unexpected transgene dependent,high-level, and widespread distribution of HSV-1 thymidine kinasethroughout the CNS, Mol. Ther., 4:490-198 (2001); A. F. Shering et al.,Cell type-specific expression in brain cell cultures from a short humancytomegalovirus major immediate early promoter depends on whether it isinserted into herpesvirus or adenovirus vectors, J. Gen. Virol.,78:445-459 (1997)]. Quality control of viral stocks was assayed bydetection of contaminating replication-competent adenovirus (RCA) [L. D.Dion et al., Supernatant rescue assay vs. polymerase chain reaction fordetection of wild type adenovirus-contaminating recombinant adenovirusstocks, J. Virol. Methods, 56:99-107 (1996)] or endotoxin (LPS), usingthe Multitest Limulus Amebocyte Lysate Pyrogen Kit (obtained fromBiowhittaker, inc.). All viral stocks used in the study were RCA and LPSfree [T. Southgate et al., Adenoviral vectors for gene transfer intoneural cells in primary culture, In: D. Sibley, Editor, CurrentProtocols in Neuroscience, Wiley, New York (2000), 4.23.1-4.23.401.

Example 4 Analysis of Shh Expression and Secretion

The expression and secretion of ShhN into the culture medium afterinfection of BHK cells with RAd-ShhN was evaluated by dot blot, Westernblot, and ELISA. edia from RAd-infected cells (i.e., CM), were preparedas described above. For dot-blot analysis, 200 μl of CM from uninfectedor RAd-ShhN or RAd-35-infected BHK cells was bound to nitrocellulosemembrane (Hybond ECL; obtained from Amersham Pharmacia Biotech) using aBio-Dot apparatus (obtained from Bio-Rad), and the nitrocellulosemembrane was probed using the 5E1 anti-ShhN monoclonal antibody (1:1000[J. Ericson et al., Two critical periods of Sonic Hedgehog signalingrequired for the specification of motor neuron identity, Cell,87:661-673 (1996)]) as primary antibody and biotinylated anti-mouseantibody (1:1000; obtained from DAKO) as secondary antibody.Colorimetric detection of Shh immunoreactivity was carried out using thebiotin-avidin-horseradish peroxidase detection kit (Vectastain ABC;obtained from Vector Laboratories). For Western blot analysis, CM fromuninfected or RAd-35 or RAd-ShhN-infected BHK cells was concentrated25-fold using an Ultrafree-0.5 centrifugal kit (obtained from Millipore)(Biomax 5-kDa NMWL-membrane). Concentrated CM samples were fractionatedby Nu-PAGE and transferred to a nitrocellulose membrane (Hybond ECL;obtained from Amersham Pharmacia Biotech) for 30 minutes at 15 V using asemidry blot transfer system (obtained from Hoeffer ScientificInstruments). The membrane was probed using a specific goat anti-ShhNpolyclonal antibody (1:100; obtained from Santa Cruz Biotechnology,Inc.) as primary antibody and a biotinylated anti-goat antibody (1:1000;obtained from DAKO) as secondary antibody. Colorimetric detection ofShhN was carried out using an ABC detection kit (obtained from VectorLaboratories). ELISA was carried out as follows: 96-well ELISA plates(obtained from Greiner) were coated with 50 μl of coating solution(coating antibody Shh 5E1 [J. Ericson et al., Two critical periods ofSonic Hedgehog signaling required for the specification of motor neuronidentity, Cell, 87:661-673 (1996)] diluted 1:500 in PBS, pH 7.2)overnight at room temperature. Coating solution was discarded the nextday. Nonspecific binding sites were blocked by incubating with 100 μl of3% BSA-PBS solution for 2 hours at 37° C. The plate was then washedthree times with 0.2% Tween 20-PBS solution. Conditioned medium fromRAd-infected BHK cells (100 μl/well) was added to the plate andincubated at 37° C. for 2 hours. After the incubation period, the platewas washed three times using a 0.1% Tween 20-PBS solution. The plate wasthen incubated with 50 μl/well of secondary antibody (goat anti-Shhpolyclonal antibody diluted 1:200 in 3% BSA-PBS solution) for 1 hour atroom temperature and washed, and 35 μl of detection antibody [anti-goatbiotinylated antibody (obtained from DAKO) diluted 1:5000 in 3% BSA-PBSsolution] was added to each well. The plate was then incubated foranother hour at room temperature and washed thoroughly to remove anyremaining unbound antibody. The plate was incubated with 100 μl of ABC(obtained from Vector Laboratories) for 30 minutes and washed. Finally,100 μl of 1 mg/ml ABTS substrate (obtained from Sigma) was added. Thereaction was developed in the dark for 30 minutes at room temperature.The presence of ShhN in conditioned medium was determined by reading theabsorbance at 405 nm.

Example 5 In Vitro Assessment of ShhN-Mediated Survival of DopaminergicMidbrain Neurons

To assay ShhN-mediated survival of dopaminergic neurons. VM cell primarycultures were plated and maintained in 50% conditioned medium frominfected and control BHK cells for 7 or 4 days, respectively. VM primaryculture cells were then fixed with 4% paraformaldehyde and 0.2 M sucrosein PBS, pH 7.4, and processed for tyrosine hydroxylase (TH)immunocytochemistry. The number of TH-positive cells present in eachcondition was counted. To confirm the specific effects of ShhN ondopaminergic neuron survival, an immunoblocking experiment was performedby adding to the conditioned medium from mock-, RAd-35-, orRAd-ShhN-treated BHK cells the 5E1 anti-ShhN antibody (to a finaldilution of 1:500 [J. Ericson et al., Two critical periods of SonicHedgehog signaling required for the specification of motor neuronidentity, Cell, 87:661-673 (1996)]) for 30 minutes at 4° C. beforeaddition of the mixture to VM cultures. VM cultures were incubated forfour further days after which they were processed for THimmunocytochemistry using the ABC kit. The number of TH-immunoreactiveneurons was counted using a total magnification of 200×. Data regardingthe survival of dopaminergic neurons in culture, as well asShhN-blocking experiments, were confirmed in three independentexperiments in triplicate, values were expressed as means±SEM, anddifferences in the survival of TH-immunoreactive neurons among thetreatments were analyzed statistically [R. Sokal, The Principles andPractice of Statistics in Biological Research, vol. 2, W.H. Freeman,Oxford (1981)]. Differences in TH+ neuronal survival were evaluated bythe Student t test. The differences between the effects of eachtreatment were assessed by one-way ANOVA. When the ANOVA showedsignificant differences, pair-wise comparisons between means were testedusing either Tukey or Dunnett multiple comparisons test [R. Sokal, ThePrinciples and Practice of Statistics in Biological Research, vol. 2,W.H. Freeman, Oxford (1981)]. Statistical tests were performed using theSPSS statistical package for Windows version 9 (obtained from SPSS,inc.).

Example 6 In Vitro Bioactivity of RAd-ShhN, RAd-Gli-1, and RAd-Nurr-1,Determined as ShhN- or Gli-1-Induced Differentiation of C3H10T1/2 Cellsor Nurr-1-Induced Luciferase Expression from a Nurr-1-ResponsivePromoter

Differentiation of the pluripotential cell line C3H10T1/2 into theosteoblastic lineage in response to RAd-ShhN or RAd-Gli-1 infection wascarried out as follows: 1×10⁵ cells per well were seeded in two six-wellplates and later infected with either RAd-ShhN or RAd-Gli-1 at m.o.i.200. Uninfected cells or a control adenovirus (RAd-35; m.o.i. 200) wasused as negative control. The induction of osteoblast phenotype inresponse to the viral treatment was determined by detecting alkalinephosphatase (AP) activity, a marker of bone differentiation, using thehistochemical detection afforded by the kit Fast Red TR/Naphtol AS-MX(obtained from Sigma). AP-positive cells (red reaction product) in eachwell were visualized under light microscopy and three independentexperiments were carried out to confirm the results. To test thebioactivity of RAd-Nurr-1, a COS-7 cell line transiently transfectedwith a plasmid containing a specific Nurr-1-responsive element, NBRE-LUC(kindly provided by Dr. J. Milbrandt), and thus responding to thepresence of Nurr-1 with an increase in luciferase activity was used.

Example 7 Detection of TH Immunoreactivity by Immunohistochemistry

Midbrain cultures or brain sections were permeabilized in 2 ml ofTBS/Triton X-100 (0.5% v/v) at room temperature and washed, andendogenous peroxidase activity was inactivated by adding 3 ml of 0.3%H₂O₂ for 15 minutes. Nonspecific antibody binding sites and Fc receptorswere blocked by incubating the sections with 10% horse serum (HS) in 1ml of TBS/Triton for 1 hour and washed, and sections were incubated witha specific rabbit anti-TH polyclonal antibody (obtained from Pharmingen)diluted 1:1000 in TBS/Triton/1% HS. This incubation was carried outovernight at room temperature. Sections were then washed and incubatedfor 4 hours with rabbit biotinylated secondary antibody (1:200) inTBS/Triton/1% horse serum. TH immunoreactivity was revealed using ABC(obtained from Vector Laboratories), and diaminobenzidinetetrahydrochloride (obtained from Sigma) was the substrate forhorseradish peroxidase. Sections were washed, mounted ontogelatin-coated slides, air-dried, dehydrated, and cover-slipped with DPXas previously described [C. E. Thomas et al., Gene transfer into ratbrain using adenoviral vectors, In: J. N. Gerfen, R. McKay, M. A.Rogawski, D. R. Sibley and P. Skolnick, Editors, Current Protocols inNeuroscience, Wiley, New York (2000). 4.23]-4.23.40].

Example 8 Immunocytochemical Detection of β-Galactosidase, HSV-1 TK,ShhN, Gli-1, and Nurr-1

The striatum or substantia nigra of animals injected with RAd-35,RAd-TK, RAd-ShhN, RAd-Gli-1, or RAd-Nurr-1 was immunostained asdescribed in detail above. ShhN was detected using a mouse monoclonalantibody raised against the amino-terminal of Shh (obtained fromUniversity of Iowa Hybridoma Bank), Gli-1 and Nurr-1 were detected usingrabbit polyclonal antibodies raised against the amino-terminal of Gli-1or against Nurr-1 (obtained from Santa Cruz Biotechnology). HSV-1 TK wasdetected with a specific polyclonal rabbit antibody raised against aTK-specific peptide and produced by the inventors and publishedelsewhere [A. J. Zermansky et al., Towards global and long-termneurological gene therapy: unexpected transgene dependent, high-level,and widespread distribution of HSV-1 thymidine kinase throughout theCNS, Mol. Ther., 4:490-498 (2001); T. D. Southgate et al., Long-termtransgene expression within the anterior pituitary gland in situ: impacton circulating hormone levels, cellular and antibody-mediated immuneresponses. Endocrinology, 142:464-476 (2001)].

Example 9 Intrastriatal Delivery of RAds in the 6-OHDA ExperimentalModel of PD and Quantification of RAd-Mediated Protection ofDopaminergic Neurons in the Substantia Nigra Pars Compacta

Adult male Fisher 344 rats of 200-250 grams body weight (obtained fromCharles River Breeding Laboratories) were used. All animals had freeaccess to food and water, a 12-hour light/dark cycle, and constanthousing temperature and humidity, and experiments followed approvedlocal regulations guiding experimental research. The ability ofRAd-ShhN, RAd-Gli-1, or RAd-Nurr-1 to protect DA neurons from 6-OHDAneurotoxicity was evaluated using a modification of a robustexperimental model of PD in rats [D. L. Choi-Lundberg et al., Behavioraland cellular protection of rat dopaminergic neurons by an adenoviralvector encoding glial cell line-derived neurotrophic factor, Exp.Neurol., 154:261-275 (1998)]. Stereotaxic neurosurgery was performed onthe animals under gaseous anesthetic as previously described [A.Hurtado-Lorenzo et al. Adenovirus for gene transfer into the rat brain:evaluation of transfer efficiency, toxicity and inflammatory and immunereactions, In: C. A. Machida, Editor, Virus Vectors for Gene Therapy:Methods and Protocols, Humana Press Inc., Totowa, N.J. (2003), 113-133],using the following stereotaxic coordinates from bregma: AP +1.0 mm, ML+3.2 mm, DV −5.0 mm for the right hemisphere injection and AP +1.0 mm,ML −3.2 mm, DV −5.0 mm for the left hemisphere injection. Using a 0.5-μlHamilton syringe, a total volume of 0.02 μl of the retrograde tracerfluoro-gold (FG) (2% diluted in saline 0.9% w/v) was injectedbilaterally, over a total of 6-7 minutes. The volume of FG was optimizedto label a number of cells comparable to those labeled in the substantianigra following the injection of RAds into the striatum. The followingvolumes of FG were tested: 0.2, 0.1, 0.05, and 0.02 μl of 2% FG. Thetracer was injected into the striatum and the total number ofretrogradely labeled striatal neurons was counted. The injection of 0.02μl of 2% FG consistently labeled a range of 30-50 neurons per25-μm-thick midbrain section throughout the nigra (a total mean of 1647neurons for all sections throughout the substantia nigra); this numberwas comparable to the number of neurons retrogradely labeled followingthe injection of RAd. Therefore, this volume of FG was chosen for theexperiments.

Following the FG injection, 3 μl of RAd (1×10⁸ IU) was injected into theright hemisphere, using a 10-μl Hamilton syringe, at the identicalcoordinates used for the first injection of FG. One week later, theanimals were prepared for a second surgery, during which 2 μl of6-OHDA-HCl, resuspended in ascorbic acid 0.2 mg/ml and diluted in saline0.9% w/v to a final concentration of 8 μg/μl, was injected over 6minutes into the right striatum in exactly the same anatomical sitepreviously injected with FG and RAd. Animals were sacrificed four weeksafter injection of 6-OHDA and the brains fixed by cardiac perfusion withoxygenated tyrode followed by 4% paraformaldehyde, pH 7.4, as previouslydescribed in detail [C. E. Thomas et al., Gene transfer into rat brainusing adenoviral vectors, In: J. N. Gerfen, R. McKay, M. A. Rogawski, D.R. Sibley and P. Skolnick, Editors, Current Protocols in Neuroscience.Wiley, New York (2000), 4.23.1-4.23.40; A. Hurtado-Lorenzo et al.,Adenovirus for gene transfer into the rat brain: evaluation of transferefficiency, toxicity and inflammatory and immune reactions, In: C. A.Machida. Editor, Virus Vectors for Gene Therapy: Methods and Protocols,Humana Press Inc., Totowa, N.J. (2003), 113-133]. Brains were post-fixedfor 6 hours at 4° C., washed with PBS, and stored in PBS containing 0.1%sodium azide at 4° C. until required. Coronal sections (25 μm thickness)of the midbrain or the forebrain (40 μm) were cut using an electronicVibratome (obtained from Leica). The midbrain was sectioned from AP−4.52 mm to AP −630 mm. The forebrain was sectioned from AP 3.20 mm toAP −1.30 mm according to the stereotactic rat brain atlas [G. Paxinos etal., The Rat Brain in Stereotaxic Coordinates (2nd ed.), Academic Press,San Diego (1986)].

The extent of neuroprotection was measured by counting, using the 20×objective, the number of FG-positive retrogradely marked nigrostriatalneurons throughout the rostrocaudal axis (AP −4.8 mm to AP −6.04) of theipsilateral (lesioned) substantia nigra pars compacta (SNpc) andexpressed as a percentage of the number of fluoro-gold-markednigrostriatal neurons in the contralateral hemisphere (unlesioned); n=7per group. The medial terminal nucleus of the accessory optic tract wasused to define the border between the SNpc and the VTA. Rats injectedwith RAd-GDNF or RAd-35 (β-galactosidase) were used as positive andnegative controls, respectively. All neurons present in all 25-μmsections cut throughout the extent of the substantia nigra were counted.Any counting method in which the dependent variable enters into thestatistical calculations (e.g., in which the percentage of neuronalsurvival and the counts are performed by someone who does not know theexperimental manipulation or potential outcomes) can be used instead ofstereology—stereology is useful mainly for unbiased estimations of verylarge numbers of cells. The operator performing the neuronal counts wasblind to the identity of the sections (i.e., from either any of thecontrol or any of the experimental groups). The estimation of thepercentage of protected susceptible neurons (PSN) was calculated usingthe following mathematical correction:

${P\; S\; N} = {\frac{{\overset{\_}{X}\mspace{14mu}\%\mspace{14mu}{protected}\mspace{14mu}{cells}} - {\overset{\_}{X}\mspace{14mu}\%\mspace{14mu}{survivor}\mspace{14mu}{cells}\mspace{14mu}\left( {{negative}\mspace{14mu}{control}} \right)}}{100 - {\overset{\_}{X}\mspace{14mu}\%\mspace{14mu}{survivor}\mspace{14mu}{cells}\mspace{14mu}\left( {{negative}\mspace{14mu}{control}} \right)}} \times 100.}$

Example 10 Quantification of the Cell Body Area of Dopamine NigralNeurons within the SNpc

The size of ipsilateral (lesioned side) dopaminergic neurons inRAd-treated animals and controls was measured as square micrometers ofcell body area and expressed as a percentage of the cell body area ofdopaminergic neurons in the contralateral site (unlesioned side). Onehundred dopaminergic neurons were randomly chosen at the level of therostral SNpc and used to estimate the area of neuronal somata; sevenanimals per group were used. The experimenter selected the neurons andwas blind to the treatment groups. Measurements were made with a LeicaQuantimet Q600 Image Analysis System controlled by QWIN software(obtained from Leica Microsystems; Cambridge, UK) connected to a LeicaRMDB microscope.

Example 11 Quantification of the Density of Striatal Dopaminergic,TH-Immunoreactive, Fibers

The extent of striatal dopaminergic denervation produced by theinjection of the neurotoxin 6-OHDA and the effect of the potentiallytherapeutic RAds was evaluated by measuring the density of TH-IR fibersin the striatum. Six representative forebrain sections corresponding tothe coordinates (from bregma) AP 1.60, AP 1.20, AP 1.00. AP 0.70, AP0.48, and AP 0.20 were used to measure the density of TH-IR in theentire ipsilateral striatum and expressed as a percentage of anequivalent area in the contralateral site (n=4). All measurements weremade with a Leica Quantimet Q600 Image Analysis System controlled byQWIN software (obtained from Leica Microsystems) connected to a LeicaRMDB microscope.

Example 12 Statistical Analysis

The treatment groups (RAd-ShhN, RAd-Gli-1. RAd-Nurr-1, RAd-35, RAd-GDNF)were compared by ANOVA or repeated-measures ANOVA with Tukey-Kramer(multiple comparisons test) or Dunnett post hoc pair-wise comparisons.Statistical calculations were made using the Graphpad Instat v2.00statistical package.

Example 13 Molecular Characterization of Recombinant Adenoviral Vectors

The shuttle vectors encoding GDNF, ShhN, Gli-1, or Nurr-1 wereco-transfected with the adenovirus 5 (Ad5) genomic plasmid pJM17 into293 cells; the structure of the expected recombinant vectors is shown inFIG. 1 a. After the onset of cytopathic effect (CPE), infected cellswere collected and their DNA was extracted to characterize therecombinant adenoviruses and confirm the presence of the transgeneswithin the adenoviral genome (RAd-GDNF, FIGS. 1 b and 1 c; RAd-ShhN,FIGS. 1 d and 1 e; RAd-Gli-1, FIGS. 1 f and 1 g; the construction ofRAd-Nurr-1 is not illustrated in detail).

FIG. 1 b shows the restriction pattern and FIG. 1 c the Southern blothybridization of the shuttle vector pALGDNF (lane 1), RAd-GDNF (lane 2),the Ad5 genomic plasmid pJM17 (lane 3), and the molecular weight (MW)markers (lane 4). DNA was extracted and digested with HindIII to releasethe insert (0.7 kb) together with the major immediate early hCMVpromoter (0.7 kb), resulting in the generation of a 1.4-kb fragment(FIGS. 1 b and 1 c) from the shuttle vector (lanes 1), and the RAd-GDNF(lanes 2), but not from pJM17 (lanes 3). Southern blot hybridization wasused, utilizing a GDNF-specific 0.7-kb DIG-labeled probe correspondingto the full-length GDNF cDNA, to identify the 1.4-kb band. As shown inFIG. 1 c the shuttle vector (lane 1) and RAd-GDNF (lane 2) exhibited theexpected 1.4-kb positive hybridization signal, whereas this signal wasabsent from the plasmid pJM17 (lane 3).

A recombinant adenovirus encoding ShhN under the control of the hCMVpromoter (RAd-ShhN) was also constructed. The viral DNA was extractedand digested with HindIII. FIG. 1 d shows the restriction patterns ofRAd-ShhN (lane 1) and the shuttle vector pALShhN (FIG. 1 d, lane 2) usedas positive control. The HindIII digestion resulted in the release of a0.9-kb fragment that corresponds to the ShhN coding region together withthe hCMV promoter (FIG. 1 d, lanes 1 and 2). The identity of the 0.9-kbband was confirmed by Southern blot hybridization using a homologousDIG-labeled probe corresponding to the ShhN cDNA. As shown in FIG. 1 e,the probe specifically hybridized with the 0.9-kb band of RAd-ShhN (FIG.1 e, lane 1) and the shuttle vector pALShhN (FIG. 1 e, lane 2).

Following the co-transfection of the shuttle vector pALGE-1 togetherwith the Ad5 genomic plasmid PJM17 into 293 cells, and the onset of CPE,the viral DNA was extracted and digested with HindIII to confirm thepresence of the Gli-1 transgene within the Ad5 genome. FIG. 1 f showsthe restriction pattern of RAd-Gli-1 in lane 1. The digestion releasedthe insert (3.6 kb) together with the poly(A) signal (0.4 kb) thatresults in the generation of a 4.0-kb fragment. The shuttle plasmidpALGli-1 digested with HindIII was used as positive control for thepresence of the expression cassette (FIG. 1 f, lane 3), and theHindIII-digested Ad5 genomic plasmid pJM17 (FIG. 1 f, lane 2) asnegative control. To confirm the identity of the 4.0-kb band a Southernhybridization was performed, using as a probe the Gli-1 cDNA fragmentlabeled with DIG. A positive hybridization signal was detected in thelane corresponding to RAd-Gli-1 (FIG. 1 g, lane 1) as well as in thelane corresponding to the shuttle vector pALGli-1 digested with HindIII(FIG. 1 g, lane 3). In contrast, no hybridization signal was detected inthe digested viral plasmid pJM17 (FIG. 1 g, lane 2) used as negativecontrol

Example 14 Release of ShhN by BHK or Primary Cultures of Glia1 CellsInfected with RAd-ShhN

Previously, it was demonstrated that by expressing the ShhN-terminalsequence, it is possible to obtain a functional and soluble peptide A.Lopez-Martinez et al., Limb-patterning activity and restricted posteriorlocalization of the amino-terminal product of Sonic hedgehog cleavage,Curr. Biol., 5:791-796 (1995); C. M. Fan et al., Long-range sclerotomeinduction by sonic hedgehog: direct role of the amino-terminal cleavageproduct and modulation by the cyclic AMP signaling pathway, Cell,81:457-465 (1995); R. B. Pepinsky et al., Identification of a palmiticacid-modified form of human Sonic hedgehog, J. Biol. Chem.,273:14037-14045 (1998); H. Roelink et al., Floor plate and motor neuroninduction by different concentrations of the amino-terminal cleavageproduct of sonic hedgehog autoproteolysis, Cell, 81:445-455 (1995); X.Zeng et al., A freely diffusible form of Sonic hedgehog mediateslong-range signalling, Nature, 411:716-720 (2001)]. To evaluate whetherRAd-ShhN expressed a soluble form of the ShhN peptide, BHK cells wereinfected with RAd-ShhN at m.o.i. 300 for 6 hours, after which thesupernatant was replaced and the cells were incubated for a further 48hours. After 48 hours, the supernatant was collected and the proteinswere concentrated. BHK cells infected with a RAd expressingβ-galactosidase (RAd-35), uninfected cells, or uninfected cells grown inthe absence of serum were used as negative controls. The presence ofShhN in the supernatant was detected by Western blot (FIGS. 2 a and 2b). A 20-kDa band corresponds to the predicted molecular weight of ShhN.This band was immunoreactive for the specific anti-Shh monoclonalantibody 5E1 in the sample obtained from RAd-ShhN-infected cells (FIG. 2b, lane 4). Such an immunoreactive band was not detected in the samplesobtained from BHK cells infected with RAd-35 (FIG. 2 b, lane 3),uninfected cells (FIG. 2 b, lane 2), or uninfected cells cultured in theabsence of serum (FIG. 2 b, lane 1).

Dot-blot analysis also further confirmed that ShhN was secreted into themedium, whereas it was not detected in the media from RAd-35-infectedcells, uninfected cells, or cells uninfected and cultured in the absenceof serum (FIG. 2 c). Finally semiquantitation of the levels of ShhNpeptide released to the medium using an ELISA technique demonstrated am.o.i.-dependent increase in secreted ShhN, reaching its peak at 300m.o.i. (FIG. 2 d).

In vivo, mostly glial cells will be infected by RAd. Thus, to determineif glial cells could produce ShhN and release it into the medium,primary cultures of glial cells were infected with increasingconcentrations of RAd-ShhN and either immunostained for ShhN (FIGS. 2 eand 2 f) or evaluated the culture medium for its content of releasedShhN (FIG. 2 g). These data showed that glial cells do express ShhN andcan release it into the medium, supporting the use of RAd-ShhN in invivo applications.

Example 15 In Vitro Bioactivity of ShhN: Conditioned Media from CulturesInfected with RAd-ShhN Promote the Survival of Dopamine Neurons inPrimary Cells

In vivo ShhN would be expected to be released and then act on dopamineneurons to exert its effects. This model was tested first in culture. Todetermine whether the ShhN peptide encoded by RAd-Shh and released frominfected cells would protect dopaminergic neurons in vitro, VM cultureswere maintained in 50% CM from BHK cells infected with RAd-ShhN(CM-ShhN) or RAd-35 (CM-35) or uninfected. VM cultures were maintainedunder stringently serum-free conditions (i.e., at no time were the cellsexposed to serum). Further, to assess the specificity of the effects ofCM-ShhN on DA neuronal survival, conditioned medium from either mock orRAd-infected BHK cells was incubated with or without a monoclonalanti-Shh blocking antibody before adding it to the VM cultures. Thecultures were kept in different CM with or without Shh-blocking antibodyfor three days and then processed for TH immunocytochemistry todetermine the effects of the treatments on dopaminergic neuron survival.

FIG. 3 illustrates the response of VM-TH+ neurons to the differenttreatments. FIGS. 3 a-3 f illustrate that the survival ofTH+-immunoreactive neurons in culture is increased only in culturestreated with CM from RAd-ShhN-infected cells (FIG. 30 and that thisincrease is blocked by pretreatment of the CM with anti-ShhN antibodies(FIG. 3 e). Further, FIG. 3 shows that control CM maintained a lowernumber of TH+-immunoreactive neurons in culture and that this effectcould not be inhibited by anti-ShhN antibodies.

The quantification of these experiments is shown in FIG. 3 g. CM fromcells infected with RAd-ShhN enhanced dopaminergic neuron survival byapprox 160%, and preincubation with anti-ShhN antibodies resulted in thecomplete inhibition of such trophic effects. This demonstrates that theincrease in DA neuronal survival in CM removed from BHK cells infectedwith RAd-ShhN is due to the presence of ShhN in the CM. In the absenceof ShhN-blocking antibodies, only the CM from BHK infected with RAd-ShhNsignificantly improved survival of TH+ neurons in VM cultures. Incontrast, in the presence of ShhN-blocking antibodies, all CM providedcomparable levels of TH+ neuronal survival, indicating that the levelsof uncharacterized BHK-derived trophic factors were similar in all CMand that these uncharacterized activities did not include ShhN.

Example 16 In Vitro Bioactivity of ShhN and Gli-1: Infection of thePluripotent Cell Line C3H10T1/2 with RAd-ShhN or RAd-Gli-1 InducesOsteoblastic Differentiation

Biochemical and genetic data suggest that the receptor for Shh is theproduct of the tumor suppressor gene patched (ptc) [J. Motoyama et al.,Ptch2, a second mouse Patched gene is co-expressed with Sonic hedgehog,Nat. Genet. 18:104-106 (1998); V. Marigo et al., Biochemical evidencethat patched is the Hedgehog receptor, Nature, 384:76-179 (1996)]. TheShh signal is received and transduced at the membrane via a receptorcomplex consisting of ptc and maw. Ptc is a 1500-amino-acid glycoproteinwith 12 membrane-spanning domains [Y. Nakano et al., A protein withseveral possible membrane-spanning domains encoded by the Drosophilasegment polarity gene patched, Nature, 341:508-513 (1989)] and twoextracellular loops that are required for Shh binding [V. Marigo et al.,Biochemical evidence that patched is the Hedgehog receptor, Nature,384:76-179 (1996); D. M. Stone et al., The tumour-suppressor genepatched encodes a candidate receptor, for Sonic hedgehog, Nature,384:129-134 (1996)]. Smo is a 115-kDa protein [J. Alcedo et al., TheDrosophila smoothened gene encodes a seven-pass membrane protein, aputative receptor for the hedgehog signal, Cell, 86:221-232 (1996)]. Inthe absence of Shh, Smo and Ptc form an inactive complex. When Shh bindsto Ptc, the complex is altered and Smo is released from inhibitorycontrol to transduce an activating signal to the nucleus [D. Kalderon,Transducing the hedgehog signal, Cell, 103:371-374 (2000)] (FIG. 4 a);this activates Gli-1, a transcription factor proposed to be a majormediator of the Shh signal [C. C. Hui et al. Expression of three mousehomologs of the Drosophila segment polarity gene cubitus interruptus,Gli, Gli-2, and Gli-3, in ectoderm-and mesoderm-derived tissues suggestsmultiple roles during postimplantation development, Dev. Biol.,162:402-413 (1994); J. Lee et Gli1 is a target of Sonic hedgehog thatinduces ventral neural tube development, Development, 124:2537-2552(1997); K. A. Platt et al., Expression of the mouse Gli and Ptc genes isadjacent to embryonic sources of hedgehog signals suggesting aconservation of pathways between flies and mice, Mech. Dev., 62:121-135(1997); H. Sasaki et al., A binding site for Gli proteins is essentialfor HNF-3beta floor plate enhancer activity in transgenics and canrespond to Shh in vitro, Development, 124:1313-1322 (1997)] (FIG. 4 a).Previous studies [N. Kinto et al., Fibroblasts expressing Sonic hedgehoginduce osteoblast differentiation and ectopic bone formation, FEESLett., 404:319-323 (1997)] demonstrated that conditioned mediumcontaining ShhN was able to induce differentiation of the pluripotentialfibroblast-like cell line C3H10T1/2 into osteoblasts, as determined bythe induction of AP activity, an early marker of bone differentiation.Based on these observations an experiment was designed to test whetherRAd-Gli-1 could replicate the morphogenetic properties of Shh. In thisexperiment 50% confluent C3H10T1/2 cells were mock infected with PBS orwith RAd-Gli-1 at an m.o.i. 200. AP activity was detected eight daysafter infection using the Fast Red Kit (obtained from Sigma). C3H10T1/2cells infected with RAd-ShhN at m.o.i. 200 were used as a positivecontrol. As shown in FIGS. 4 b and 4 c, infection with RAd-Gli-1 andRAd-ShhN was able to induce differentiation of the pluripotent cell lineC3H10T1/2 into osteoblasts as inferred from the detection of APactivity. As expected from a protein targeted to the secretory pathway,ShhN immunoreactivity outlined the ER/Golgi compartments (FIG. 4 b).Gli-1 has been shown to shuttle between the cytoplasm and the nucleus.Thus, the immunoreactivity detected in both the cytoplasm and thenucleus is expected from previous knowledge of the subcellulardistribution and function of Gli-1 (FIG. 4 b; note that due to thedifferent levels of expression in either compartment the figure showshigher cytoplasmic localization). In contrast, cells incubated with PBSdid not induce osteoblast differentiation of this cell line. Theseresults demonstrated that RAd-Gli-1 encodes a transcription factor thatis biologically active, and, more importantly, that it is able to mimicthe effects of ShhN in vitro.

Example 17 In Vitro Bioactivity of RAd-Nurr-1

The bioactivity of RAd-Nurr-1 was tested using COS-7 cells transientlytransfected with a reporter plasmid containing the Nurr-1-responsiveelement NBRE upstream of the prolactin promoter (Pro36), as illustratedin FIG. 4 d. Expressed Nurr-1 binds to the NBRE and stimulatesPro36-driven luciferase expression. Infection of transfected COS-7 cellswith RAd-Nurr-1 increased luciferase expression two to three times overcontrol values. Uninfected cells, or cells infected with a vectorexpressing the antisense noncoding strand of Nurr-1, did not induceluciferase expression over the basal activity of the reporter constructeven in the absence of infection with RAd-Nurr-1 (FIG. 4 e).Immunohistochemistry of control COS-7 cells infected with RAd-Nurr-1indicates strong immunoreaction for Nurr-1 in the nucleus of infectedcells (FIG. 4 f).

Example 18 Distribution of Transgenes Throughout the Rostrocaudal Extentof the Substantia Nigra Following the Injection of RAds into theStriatum

To determine the distribution throughout the rostrocaudal extent of thesubstantia nigra of an intracellular transgene expressed from an RAd,RAd-TK was injected into the striatum and the immunocytochemicaldistribution of the transgene was assessed in retrogradely labeledneurons in the substantia nigra. RAd-TK encodes the full-length herpessimplex virus type 1 thymidine kinase, HSV-1 TK [R. A. Dewey et al.,Chronic brain inflammation and persistent herpes simplex virus 1thymidine kinase expression in survivors of syngeneic glioma treated byadenovirus-mediated gene therapy: implications for clinical trials, Nat.Med., 5:1256-1263 (1999); A. J. Zermansky et al., Towards global andlong-term neurological gene therapy: unexpected transgene dependent,high-level, and widespread distribution of HSV-1 thymidine kinasethroughout the CNS, Mol. Ther., 4:490-498 (2001)]. The wide distributionthroughout the rostrocaudal extent of the transgene TK detectedimmunocytochemically throughout the substantia nigra is illustrated inFIG. 5.

Example 19 Combined Retrograde Targeting of Substantia NigraDopaminergic Neurons with Both Fluoro-Gold and Adenoviral Vectors

FIG. 6 illustrates the injection site and distribution of fluoro-gold(green) in the striatum, as well as the site of injection of RAd,detected by immunocytochemistry for the transgene (red) (FIG. 6 a). Theoverlap between both labels indicates that fluoro-gold and RAd havedistributed over an equivalent area of striatal tissue. FIG. 6 b showsthe detection of retrogradely transported fluoro-gold in neurons alsoexpressing a RAd-encoded transgene. This demonstrates that RAd-encodedtherapeutic transgene expression occurs in nigral neurons that projectto the striatum. Finally, FIGS. 6 c-6 g indicate that all neuronscontaining the retrogradely transported dye fluoro-gold are TH+ nigralneurons and thus identifies these as bona fide dopaminergicnigrostriatal neurons.

Example 20 RAd-ShhN and RAd-Gli-1, but not RAd-Nurr-1 ProtectDopaminergic (DA) Nigrostriatal Neurons Against 6-OHDA-InducedNeurodegeneration In Vivo

The ability of RAd-Shh and RAd-Gli-1 to protect DA neurons from 6-OHDAneurotoxicity was evaluated using a modification of the rat model of PDreported by Choi-Lundberg [D. L. Choi-Lundberg et al., Behavioral andcellular protection of rat dopaminergic neurons by an adenoviral vectorencoding glial cell line-derived neurotrophic factor. Exp. Neurol.,154:261-275 (1998)] (illustrated in FIG. 6). Fluoro-gold was injectedintrastriatally on both sides of the brain; during the same surgicalintervention 1×10⁸ IU of RAd-ShhN, RAd-Gli-1, RAd-Nurr-1, the negativecontrol vector RAd-35, or the positive control RAd-GDNF was injectedinto the right striatum. One week later, retrograde degeneration of thenigrostriatal pathway was induced by unilateral (right dorsal striatum)administration of 16 μg of 6-OHDA-HCl using the same coordinates usedfor the delivery of fluoro-gold and RAd (referred to as the “ipsilateralsite”, and four weeks later, the rats were injected with an overdose ofanesthetic, were perfused-fixed through the left ventricle of the heart,were postfixed overnight, and 25-μm-thick sections were cut on avibratome and analyzed using an Olympus AHBS fluorescencephotomicroscope. The surviving neurons were counted on the ipsilateralside (exposed to the neurotoxin) and expressed as a percentage ofneurons of the contralateral (control) hemisphere.

Animals injected with the negative control vector RAd-35 preserved only33.4±1.83% of nigrostriatal neurons (FIGS. 7 a, 7 b, and 7 i). Neuronalcounts indicated that throughout the SNpc of rats injected withRAd-ShhN, 53.3±1.29% of dopaminergic neurons survived on the lesionedsite in comparison with the intact contralateral site (FIGS. 7 e, 7 f,and 7 i). In animals injected with RAd-Gli-1 52.9±3.31% offluoro-gold-labeled neurons survived (FIGS. 7 g, 7 h, and 7 i), while79±3.7% of retrogradely labeled dopaminergic neurons were protected fromneurodegeneration by RAd-GDNF (FIGS. 7 c, 7 d, and 7 i). Animalsinjected with RAd-Nurr-1 retained only 27±1.22% of labeled striatonigralneurons, a number not statistically different from that of animalsinjected with the negative control vector RAd-35 (FIG. 7 i). Theseresults demonstrate that intrastriatal delivery of 1×10⁸ IU of RAd-ShhNand RAd-Gli-1 can protect a statistically significant proportion ofnigrostriatal neurons susceptible to being killed by 6-OHDA [analysis ofvariance (ANOVA), F=38.33, P≦0.01, n=7]; RAd-GDNF protected a higherproportion of nigrostriatal neurons (ANOVA. F=38.33, P≦0.001, n=7),while RAd-Nurr-1, however, was ineffective.

Example 21 RAd-ShhN or RAd-Gli-1 does not Protect Dopamine Neurons'Tyrosine-Hydroxylase-Immunoreactive Terminals in the Striatum: aComparison with RAd-GDNF

Having demonstrated that a single injection of 1×10⁸ IU of RAd-Shh andRAd-Gli-1 is neuroprotective for retrogradely labeled dopaminergicneurons in the SNpc, it was examined whether these vectors could prevent6-OHDA-induced dopaminergic denervation of the striatum. Forebraincoronal sections were processed from animals injected with 1×10⁸ IU ofRAd-ShhN or RAd-Gli-1 by immunohistochemistry to detectTH-immunoreactive (TH-IR) fibers in the striatum. The extent of striataldenervation produced by 6-OHDA was evaluated by measuring the density ofTH-IR fibers in the entire ipsilateral striatum; this was expressed as apercentage of the contralateral site. The density of TH-IR fibers in thestriatum of rats treated with RAd-ShhN and RAd-Gli-1 decreased to42.4±1.26 and 50.62±4.25 of the contralateral site, respectively (FIG.8). In rats treated with RAd-35, the density of TH-IR fibers decreasedto 40±0.25% of controls, while in rats treated with RAd-GDNF, theydecreased only to 64%±6.72 of control values (FIG. 8).

Statistical analysis of these data indicated that neither RAd-Shh norRAd-Gli-1 was able to protect striatal axonal DA terminals fromdegeneration four weeks after injection of 6-OHDA (ANOVA. F=25.22,P≧0.05, n=4). Only the injection of RAd-GDNF resulted in a statisticallysignificant increase (ANOVA. F=25.22, P≦0.01, n=4) in the density ofTH-IR in comparison with RAd-35-treated rats. These results indicatethat while RAd-mediated gene transfer of ShhN and Gli-1 results in theprotection of DA neurons from 6-OHDA neurotoxicity, denervation ofstriatal DA terminals is not prevented.

Example 22 RAd-ShhN or RAd-Gli-1 Partially Protects DopamineStriatonigral Neuronal Cell Bodies from Atrophy Induced by IntrastiatalInjection of the Neurotoxin 6-OHDA

Although not wishing to be bound by any particular theory, the inventorshypothesized that if GDNF could protect the striatal dopaminergicinnervation, it could also possibly protect the decrease in cell bodysize of dopaminergic neurons in the substantia nigra that is caused bydopaminergic denervation, with cells with larger terminal fieldsdisplaying larger sizes and vice versa. To test this hypothesis, theinventors measured the area occupied by nigrostriatal cell bodies (n=100TH+ neurons on both the control and the neuroprotected substantia nigra)and expressed values of ipsilateral DA neuronal cell body area(neuroprotected) as a percentage of the cell body area of neurons in thecontralateral substantia nigra (FIG. 7 j). In rats injected withRAd-GDNF, cell body size decreased to 92±1.26% of the contralateralsite. In contrast, in RAd-ShhN- and RAd-Gli-1-treated rats the cell bodyarea of nigrostriatal neurons was reduced to 82% of the contralateralside neurons. Soma size in rats injected with the control vector RAd-35decreased to 67±5.24% of the contralateral side. The reduction in sizeof dopaminergic neurons in rats treated with RAd-ShhN and RAd-Gli-1 wasstatistically significant (F=51.49, P≦0.01, n=100 dopaminergic neuronsanalyzed in total or F=14.054, P≦0.05, n=7 when the data are analyzedper number of animals studied) compared with the size of those inanimals injected with RAd-GDNF. The reduction in cell body size inanimals injected with the control vector RAd-35 was statisticallysignificant compared with RAd-ShhN-, RAd-Gli-1-(ANOVA, F=51.49, P≦0.01,n=100 or F=14.054, P≦0.05, n=7), and RAd-GDNF-treated rats (ANOVA.F=51.49, P≦0.001, n=100 or F=14.054, P≦0.001, n=7).

These results indicate that, despite comparable striatal denervationseen in animals injected with 6-OHDA and treated with either RAd-ShhNand RAd-Gli-1 or the control vector RAd-35, only RAd-ShhN and RAd-Gli-1treatment partially prevented the progressive decrease of nigrostriatalcell body size induced by the neurotoxin. This indicates that ShhN andGli-1 can protect the size of dopamine neurons in the substantia nigra,independent from trophic effects at the level of the striatum and/or thestriatal axonal terminals.

Example 23 Expression of ShhN and Gli-1 in the Substantia Nigra at 1 and4 Weeks Following their Injection into the Dorsal Striatum

To confirm that the transgenes were present throughout the substantianigra during the experimental procedure, the expression of ShhN andGli-1 was determined in the substantia nigra at one and four weeks aftertheir intrastriatal injection. Using specific immunohistochemicaltechniques, both transgenes could be detected in the substantia nigra atone or four weeks after the injection of viruses into the striatum (FIG.9). This indicates that the potential neurotrophic factors wereavailable in the substantia nigra at the proper time to exert theirpharmacological effects. There was no positive immunoreaction in thecontralateral substantia nigra (not shown). Immunoreactivity for Nurr-1was present, but it was also present in many other areas of the ratbrain, including the contralateral substantia nigra (not shown). Thus,due to the high levels of basal expression of Nurr-1 an increase due toRAd-Nurr-1 expression could not be detected.

While the description above refers to particular embodiments of thepresent invention, it should be readily apparent to people of ordinaryskill in the art that a number of modifications may be made withoutdeparting from the spirit thereof. The accompanying claims are intendedto cover such modifications as would fall within the true spirit andscope of the invention. The presently disclosed embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than the foregoing description. All changes that comewithin the meaning of and range of equivalency of the claims areintended to be embraced therein.

What is claimed is:
 1. A method for treating Parkinson's disease in amammal, comprising: providing a composition comprising a Gli-1 protein;and administering a therapeutically effective amount of the compositionto the mammal.
 2. The method of claim 1, wherein the composition furthercomprises a pharmaceutically acceptable carrier.
 3. The method of claim1, wherein the mammal is human.
 4. The method of claim 1, wherein thetherapeutically effective amount of the composition is administered tothe mammal via direct injection.